Regulating Stem Cells

ABSTRACT

A composition of matter is provided, comprising a population of cultured cells that comprises a sub-population of cells that both stain as CD31Bright and demonstrate uptake of Ac-LDL+, and secrete IL-8. A method is also provided, comprising stimulating in vitro an initiating cell population (ICP) of at least 5 million cells that have a density of less than 1.072 g/ml, wherein at least 1% of the cells of the ICP is CD34+CD45−/Dim, and at least 25% of the cells of the ICP are CD31Bright, to differentiate into a progenitor/precursor cell population (PCP). Other embodiments are also described.

CROSS-REFERENCES TO RELATED APPLICATIONS

The present application claims priority from U.S. Provisional PatentApplication 60/780,781 to Porat et al., filed Mar. 8, 2006, entitled,“Regulating stem cells,” which is assigned to the assignee of thepresent invention and is incorporated herein by reference.

FIELD OF THE INVENTION

The present invention generally relates to regulating stem cells.Specifically, the present invention relates to the induction ofmigration and differentiation of stem cells.

BACKGROUND OF THE INVENTION

Since the discovery of stem cells, it has been understood that they havesignificant potential to effectively treat many diseases [1].Pluripotent stem cells derived from embryos and fetal tissue have thepotential to produce more than 200 different known cell types, and thuscan potentially replace dying or damaged cells of any specific tissue[2, 3]. Stem cells differ from other types of cells in the body, and,regardless of their source, have three general properties: (a) they arecapable of dividing and renewing themselves for long periods, (b) theyare undifferentiated, and (c) they can give rise to specialized celltypes.

Stem cells have been identified in most organs and tissues, and can befound in adult animals and humans. Committed adult stem cells (alsoreferred as somatic stem cells) were identified long ago in bone marrow.In the past decade, committed adult stem cells have also been identifiedin tissues that were previously not thought to contain them, such asbrain tissue, skin tissue, and skeletal muscle tissue [8, 9, 10, 11, 12,13]. It was initially believed that adult stem cells aretissue-committed cells that can only differentiate into cells of thesame tissue and thus regenerate the damaged tissue [1, 4, 5, 6, 7].However, recent work suggests that adult organ-specific stem cells arecapable of differentiating into cells of different tissues [8, 9, 10,11, 14, 16]. Transplantation of cells derived from brain, muscle, skinand fat tissue has been shown to result in a detectable contribution inseveral lineages distinct from their tissue of origin [8, 9, 10, 11].For example, recent reports support the view that cells derived fromhematopoietic stem cells (HSCs) can differentiate into cells native tothe adult brain [14], providing additional evidence for the plasticityof such stem cells.

The HSC is the best characterized stem cell. This cell, which originatesin bone marrow, peripheral blood, cord blood, the fetal liver, and theyolk sac, generates blood cells and gives rise to multiple hematopoieticlineages. As early as 1998, researchers reported that pluripotent stemcells from bone marrow can, under certain conditions, develop intoseveral cell types different from known hematopoietic cells [13, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27]. Such an ability to change lineageis referred to as cellular transdifferentiation or cell plasticity. Bonemarrow-derived stem cells (BMSCs) have already been shown to have theability to differentiate into adipocytes, chondrocytes, osteocytes,hepatocytes, endothelial cells, skeletal muscle cells, and neurons [28,29, 30, 31, 32].

The process of stem cell differentiation is controlled by internalsignals, which are activated by genes within the cell, and by externalsignals for cell differentiation that include chemicals secreted byother cells, physical contact with neighboring cells, and certainmolecules in the microenvironment [33, 34]. For example, if embryonicstem cells are allowed to aggregate to form embryoid bodies, they beginto differentiate spontaneously. Embryonic cells of embryoid bodies canform muscle cells, nerve cells, and many other cell types [35, 36].Although spontaneous differentiation is a good indication that a cultureof embryonic stem cells is healthy, it is not an efficient way toproduce cultures of specific cell types. In order to generate culturesof specific types of differentiated cells, e.g., myocytes, blood cells,or nerve cells, scientists must control the multiplication and thedifferentiation of stem cells by regulating the chemical composition ofthe culture medium, altering the surface of the culture dish, and/or byinserting specific genes.

Successful attempts have been made in vitro to induce differentiation ofadult stem cells into other cells by co-culturing with other adultcells. For example, recent work has shown that co-culturing adult mouseBMSCs and embryonic heart tissue causes the BMSCs to both integrate intocardiac tissue and differentiate into cardiomyocytes (CMCs). Other workhas shown that mesenchymal stem cells acquire characteristics of cellsin the periodontal ligament when co-cultured with periodontal ligamenttissue [37, 38].

Tissue injury may be one of the stimulants for the recruitment of stemcells to an injured site, by causing changes in the tissue environment,thereby drawing stem cells from peripheral blood, as well as triggeringtissue replacement by locally resident stem cells. Some reports ofelevated levels of chemokines and chemokine receptors such as CXCR4-SDFexplain some of this in vivo stem cell recruitment [39]. Other reportssuggest an important role of the chemokine CXCR8 (IL-8) as ananti-apoptotic agent which promotes tissue survival and inducesrecruitment of endogenous stem/progenitor cells [M, N, O]. An example ofthis mechanism can be seen in recent work showing that stem cellsdifferentiate into liver cells when co-cultured with injured liver cellsseparated from the stem cells by a barrier [30].

CD31, the platelet endothelial cell adhesion molecule-1 (PECAM-1), iswidely used as a marker during the development of endothelial cellprogenitors, vasculogenesis and angiogenesis (A, B, C, D, E, F, H1).CD31 is constitutively expressed on the surface of adult and embryonicendothelial cells, is a major constituent of the endothelial cellintercellular junction (where up to 10̂6 PECAM-1 molecules areconcentrated) and is weakly expressed on many peripheral leukocytes andplatelets (E, G, H). With a few minor exceptions, CD31 is not present onfibroblasts, epithelium, muscle, or other nonvascular cells.Independently of CD31 expression, endothelial cells and theirprogenitors are typically characterized by binding of Ulex-lectin incombination with the ability to uptake Acetylated-Low DensityLipoprotein (Ac-LDL) (I).

Regenerative medicine is an emerging scientific field with implicationsfor both basic and practical research. Stem and progenitor cells areapplied in a form of cellular therapy for local tissue repair andregeneration [41, 42]. These treatments aim to treat disorders inpractically all tissues and organs, such as the bladder, intestine,kidney, trachea, eye, heart valves, and bones [43, 44]. Intensivestudies are being conducted worldwide in order to generate stemcell-based tissue engineering therapies. These studies includeexperiments for the regeneration of blood vessels [13], bone [35, 45],cartilage, cornea, dentin, heart muscle [46], liver, pancreas [47],nervous tissue, skeletal muscle, and skin [18, 34, 48, 49]. Stemcell-based therapies can use cells from various organs in order togenerate different tissues. For example, epithelial surfaces (taken fromvarious tissues such as the skin, cornea and mucosal membrane) may beused as a source for corneal and skeletal tissues [50, 51].Additionally, in a more widespread application, blood marrow-derivedstern cells are used for regeneration of several different tissues suchas bone, cartilage, adipocytes, neurons, and cells of the hematopoieticsystem [33, 42].

Stem cells can be administrated systemically or locally using injectionsto the injured site. However, other potential administration routes andusage of different medical devices are being developed and tested.Different medical devices such as chemical, metal or biodegradable baseddevices have been described for the administration of stem cells intothe heart and blood vessels (J, K).

US Patent Application Publication 2004/0228847 to Goldschmidt-Clermontet al., which is incorporated herein by reference, describesstem/progenitor cells and, in particular, therapeutic strategies basedon the use of such cells to effect vascular rejuvenation and/or to serveas delivery vehicles.

PCT Patent Publication WO 2005/120090 to Fulga et al., which is assignedto the assignee of the present patent application and is incorporatedherein by reference, describes a method for use with extracted blood,including (a) applying blood to a first gradient suitable for selectingfirst-pass cells having a density less than 1.077 g/ml; (b) applying thefirst-pass cells to a second gradient suitable for selecting second-passcells having a density between 1.055 and 1.074 g/ml; (c) increasing thenumber of cells having a density between 1.055 and 1.074 g/ml, byculturing the second-pass cells for a period lasting between 3 and 30days; and (d) identifying endothelial progenitor cells in the culturedcells. Other embodiments are also described.

United States Patent Application Publication 2004-0228897 to Zhang etal., which is incorporated herein by reference, describes a medicaldevice for use to assist stem cell and/or stem cell derivatives inrepopulating, repairing and/or replacing the heart tissue in a failingheart muscle, in order to restore the heart's ability to pump blood. Themedical device is made of biocompatible materials. The specific designof the device is described as facilitating the stem cells coated in thedevice to repopulate heart muscles inside the heart. Stem cells areattached to the coated device, proliferated and/or differentiated on thedevice in a bioreactor before implantation. The device also containsbioactive components that diminish rejection by the host's immunesystem. The device may be directly implanted into the failing heartmuscle area to assist stem cells to repair failing heart muscles viasurgical and/or percutaneous catheter based procedures. In anotherembodiment, the device may be implanted to the surgical site whereabnormal heart muscles are removed, to assist stem cells to repopulateheart muscles, to replace the failing heart muscles.

US Patent Application Publication 2005/0209556 to Tresco et al., whichis incorporated herein by reference, describes a device and method forthe delivery of cells, tissues, enzymes and/or pharmacological agentsfor the treatment or prevention of diseases, disorders or deficiencies.The device is placed intravascularly and includes a chamber that housesliving cells delimited by a membrane on either side that physicallyseparates the cells from the blood stream and the central lumen of thecatheter. The device can be inserted over a guidewire and permitsflushing and reloading of the central lumen with viability supportingfactors that sustain the cells in the outer chamber for long indwellingtimes without removing it from the body. In addition, the central lumencan be used to deliver therapeutic substances or withdraw blood. The newintravascular catheter is described as being able to be used for thetreatment or prevention of a variety of diseases and disorders, and mayuse the implantation of living cells, tissues, enzymes orpharmacological agents. The device is described as being used, forexample, for non-therapeutic purposes that may involve sustainedintravascular release of biological factors as, for example, instimulating growth of farm animals to augment the production of meat.Placement of cells within the device for release of angiogenesis,cytokines, enzymes, and other factors is described. The use of stemcells within the device is also described.

U.S. Pat. No. 6,810,286 to Donovan et al., which is incorporated hereinby reference, describes a stimulatory device for the controlledproduction of angiogenic growth factors. More specifically, asubthreshold pulse generator is used for the local production ofvascular endothelial growth factor.

The following references, which are incorporated herein by reference,may be of interest:

1. Leblond C. P. (1964), “Classification of cell populations on thebasis of their proliferative behaviour,” Natl. Cancer Inst. Monogr.14:119-150

2. Evans M. J. and Kaufman M. H. (1981), “Establishment in culture ofpluripotential cells from mouse embryos,” Nature 292:154-156

3. Donovan P. J. and Gearhart J. (2001), “The end of the beginning forpluripotent stem cells,” Nature 414:92-97

4. Spradling A. et al. (2001), “Stem cells find their niche,” Nature414:98-104

5. Weissman I. L. et al. (2001), “Stem and progenitor cells: origins,phenotypes, lineage commitments, and transdifferentiations,” Annu. Rev.Cell. Dev. Biol. 17:387-403

6. Weissman I. L. (2000), “Stem cells: units of development, units ofregeneration, and units in evolution,” Cell 100:157-68

7. Cheng A, Wang S, Cai J, Rao M S, Mattson M P (2003), “Nitric oxideacts in a positive feedback loop with BDNF to regulate neural progenitorcell proliferation and differentiation in the mammalian brain,” DevBiol. 258(2):319-33

8. Cousin B, Andre M, Arnaud E, Penicaud L, Casteilla L (2003),“Reconstitution of lethally irradiated mice by cells isolated fromadipose tissue,” Biochem Biophys Res Commun. 301(4):1016-22

9. Anderson D. J., Gage, F. H., and Weissman, I. L. (2001), “Can stemcells cross lineage boundaries?” Nat. Med. 7:393-395

10. Robey P. G. (2000), “Stem cells near the century mark,” J. Clin.Invest. 105:1489-1491

11. Eisenberg L M, Burns L, Eisenberg C A (2003), “Hematopoietic cellsfrom bone marrow have the potential to differentiate into cardiomyocytesin vitro,” Anat Rec. 274A(1):870-82

12. Karl J. L., Fernandes Ian A. McKenzie, Pleasantine Mill et al.(2004), “A dermal niche for multipotent adult skin-derived precursorcells,” Nature Cell Biology Published online: 1 Nov. 2004, DOI:10.1038/ncb1181

13. Jackson K A, Mi T, Goodell M A (1999), “Hematopoietic potential ofstem cells isolated from murine skeletal muscle,” Proc Natl Acad Sci USA96(25):14482-6

14. Brazelton T R, Rossi F M, Keshet G I, Blau H M (2000), “From marrowto brain: expression of neuronal phenotypes in adult mice,” Science290(5497):1775-9

15. Bjornson C R, Rietze R L, Reynolds B A, Magli M C, Vescovi A L(1999), “Turning brain into blood: a hematopoietic fate adopted by adultneural stem cells in vivo,” Science 283(5401):534-7

16. Slack, J. M. (2000), “Stem cells in epithelial tissues,” Science287:1431-1433

17. Ferrari G., Cusella-De Angelis G., Coletta M., Paolucci E.,Stornaiuolo A., Cossu G., and Mavilio F. (1998), “Muscle regeneration bybone marrow-derived myogenic progenitors,” Science 279:528-30

18. Lagasse E, Connors H, Al-Dhalimy M, Reitsma M, Dohse M, Osborne L,Wang X, Finegold M, Weissman I L, Grompe M (2000), “Purifiedhematopoietic stem cells can differentiate into hepatocytes in vivo,”Nat Med. 6:1229-34

19. Hirschi, K. K., and Goodell, M. A. (2002), “Hematopoietic, vascularand cardiac fates of bone marrow-derived stem cells,” Gene Ther.9:648-652

20. Theise N. D. et al. (2000), “Liver from bone marrow in humans,”Hepatology 32:11-16

21. Kleeberger W. et al. (2002), “High frequency of epithelial chimerismin liver transplants demonstrated by microdissection and STR-analysis,”Hepatology 35:110-116

22. Weimann J. M. et al. (2003), “Contribution of transplanted bonemarrow cells to Purkinje neurons in human adult brains,” Proc. Natl.Acad. Sci. USA 100:2088-2093

23. Quaini F. et al. (2002), “Chimerism of the transplanted heart,” N.Engl. Med 346:5-15

24. Blau H. M. et al. (2001), “The evolving concept of a stem cell:entity or function?” Cell 105:829-841

25. Goodell M. A. et al. (2001), “Stein cell plasticity in muscle andbone marrow,” Ann. NY Acad. Sci. 938:208-218

26. Krause D. S. (2002), “Plasticity of marrow-derived stem cells,” GeneTher. 9:754-75 8

27. Wulf G. G. et al. (2001), “Somatic stein cell plasticity,” ExpHematol. 29:1361-1370

28. Pittenger M. F. et al. (1999), “Multilineage potential of adulthuman mesenchymal stem cells,” Science 284:143-147

29. Liechty K. W. et al. (2000), “Human mesenchymal stem cells engraftand demonstrate site-specific differentiation after in uterotransplantation in sheep,” Nature Med. 6:1282-1286

XIII. Li A. et al. (2003), “IL-8 directly enhanced endothelial cellsurvival, proliferation, and matrix metalloproteinases production andregulated angiogenesis”, Journal of immunol. 170:3369-3376.

XIV. Laterveer L. et al. (1995), “Interleukin-8 induces rapidmobilization of hematopoietic stem cells with radioprotective capacityand long-term myelolymphoid repopulating ability”, Blood 85:2269-75.

XV. Scho″mig K. et al. (2006), “Interleukin-8 is associated withcirculating CD133+ progenitor cells in acute myocardial infarction”,European Heart Journal 27: 1032-1037

30. Sang Y Y, Collector M I, Baylin S B, Diehl A M, Sharkis S J (2004),“Hematopoietic stem cells convert into liver cells within days withoutfusion,” Nat Cell Biol. 6(6):532-9. Epub May 9, 2004

31. Bittner R. E., Schofer C., Weipoltshammer K., Ivanova S., StreubelB., Hauser E., Freilinger M., Hoger H., Elbe-Burger A., and Wachtler F.(1999), “Recruitment of bone-marrow-derived cells by skeletal andcardiac muscle in adult dystrophic mdx mice,” Anat. Embryol. (Berl)199:391-396

32. Mezey E, Chandross K J, Harta G, Maki R A, McKercher S R (2000),“Turning blood into brain: cells bearing neuronal antigens generated invivo from bone marrow,” Science. 290(5497):1779-82

33. Douglas W. L., Dimmeler S. (2004), “Therapeutic angiogenesis andvasculogenesis for ischemic diseases. Part I: Angiogenic cytokines,”Circulation 109:2487-2491

34. Douglas W. L., Dimmeler S. (2004), “Therapeutic angiogenesis andvasculogenesis for ischemic diseases. Part II: Cell-based therapy,”Circulation 109:2692-2697

35. Rodda S J, Kavanagh S J, Rathjen J, Rathjen P D (2002), “Embryonicstem cell differentiation and the analysis of mammalian development,”Int J Dev Biol. 46(4):449-58

36. Amit M., Carpenter M. K., Inokuma M. S., Chiu C. P., Harris C. P.,Waknitz M. A., Itskovitz-Eldor J., and Thomson J. A. (2000), “Clonallyderived human embryonic stem cell lines maintain pluripotency andproliferative potential for prolonged periods of culture,” Dev Biol.227(2):271-8

37. Aoki S, Toda S, Sakemi T, Sugihara H (2003), “Coculture ofendothelial cells and mature adipocytes actively promotes immaturepreadipocyte development in vitro,” Cell Struct Funct. 28(1):55-60

38. Wan H, An Y, Zhang Z, Zhang Y, Wang Z (2003), “Differentiation ofrat embryonic neural stem cells promoted by co-cultured Schwann cells,”Chin Med J (Engl). 116(3):428-31

39. Kollet O, Shivtiel S, Chen Y Q. et al. (2003), “HGF, SDF-1, andMMP-9 are involved in stress-induced human CD34+ stem cell recruitmentto the liver,” J Clin Invest. 112(2):160-9

40. Badorff C, Brandes R P, Popp R, Rupp S, Urbich C, Aicher A, FlemingI, Busse R, Zeiher A M, Dimmeler S (2003), “Transdifferentiation ofblood-derived human adult endothelial progenitor cells into functionallyactive cardiomyocytes,” Circulation 107(7):1024-32

41. Bianco, P. and Robey P. G. (2001), “Stem cells in tissueengineering,” Nature 414:118-121

42. Lagasse E. et al. (2001), “Toward regenerative medicine,” Immunity14:425-436

43. Stock U. A., Vacanti J. P. (2001), “Tissue engineering: currentstate and prospects,” Ann. Rev. Med 52:443-451

44. Kim W. S. et al. (1994), “Bone defect repair with tissue-engineeredcartilage,” Plast. Recontr. Surg. 94:580-584

45. Petite H. et al. (2000), “Tissue-engineered bone regeneration,”Nature Biotechnol. 18:959-963

46. Jackson K A, Majka S M, Wang H, Pocius J, Hartley C J, Majesky M W,Entman M L, Michael L H, Hirschi K K, Goodell M A (2001), “Regenerationof ischemic cardiac muscle and vascular endothelium by adult stemcells,” J Clin Invest. 107(11):1395-402

47. Ramiya V. K. et al. (2000), “Reversal of insulin-dependent diabetesusing islets generated in vitro from pancreatic stem cells,” NatureMedicine 6:278-282

48. Rafii S., Lyden D. (2003), “Therapeutic stem and progenitor celltransplantation for organ vascularization and regeneration,” NatureMedicine 9:702-712

49. Gussoni E., Soneoka Y., Strickland C., Buzney E., Khan M., Flint A.,Kunkel L., and Mulligan R. (1999), “Dystrophin expression in the mdxmouse restored by stem cell transplantation,” Nature 401:390-4

50. Zhao Y et al. (2003), “A human peripheral blood monocyte-derivedsubset acts as pluripotent stem cells,” Proc. Natl. Acad. Sci. USA100:2426-2431

51. Kohji N, Masayuki Y, Yasutaka H. et al. (2004), “Cornealreconstruction with tissue-engineered cell sheets composed of autologousoral mucosal epithelium,” N Engl J Med 351:1187-96

52. Kayisli U. A., Luk J, Guzeloglu-Kayisli O. et al. (2005),“Regulation of angiogenic activity of human endometrial endothelialcells in culture by ovarian steroids,” J Clin Endocrinol Metab89:5794-5802

53. Dimmeler S. (2005), “Circulating endothelial precursors:Identification of functional subpopulations,” Blood 106(7):2231-2232

54. Urbich C. et al. (2004), “Endothelial progenitor cells:Characterization and role in vascular biology,” Circulation Research95:343-353

A. Asahara T, Murohara T, Sullivan A, et al. (1997), “Isolation ofputative progenitor endothelial cells for angiogenesis,” Science275:964-967.

B. Kalka C, Masuda H, Takahashi T, et al. (2000), “Transplantation of exvivo expanded endothelial progenitor cells for therapeuticneovascularization,” Proc Natl Acad Sci USA. 97:3422-3427.

C. Assmus B, Schachinger V, Teupe C, et al. (2002), “Transplantation ofProgenitor Cells and Regeneration Enhancement in Acute MyocardialInfarction (TOPCARE-AMI),” Circulation. 106:3009-3017.

D. Yoon C. H, Hur J., Park K W, et al. (2005), “SynergisticNeovascularization by Mixed Transplantation of Early EndothelialProgenitor Cells and Late Outgrowth Endothelial Cells,” Circulation.112:1618-1627.

K DeLisser, H. M., Christofidou-Solomidou, R. M. Strieter, M. D. et al.(1997), “Involvement of endothelial PECAM-1/CD31 in angiogenesis,” Am JPathol. 151: 671 - 677.

F. Kawamoto A., Tkebuchava T., Yamaguchi J. I., et al. (2003),“Intramyocardial Transplantation of Autologous Endothelial ProgenitorCells for Therapeutic Neovascularization of Myocardial Ischemia,”Circulation. 107:461-468

G. Newman P. J. (1997), “The Biology of PECAM-1,” J Clin Invest. 99:3-8.

H. Vecchi, A., C. Garlanda, M. G. Lampugnani, M. Resnati, et al. (1994),“Monoclonal antibodies specific for endothelial cells of mouse bloodvessels. Their application in the identification of adult and embryonicendothelium,” Eur J Cell Biol. 63: 247 - 254.

H1. Kanayasu-Toyoda T., Yamaguchi T., Oshizawa T., et al. (2003), “CD31(PECAM-1)-Bright Cells Derived From AC133-Positive Cells in HumanPeripheral Blood as Endothelial-Precursor Cells,” Journal of Cell.Physiol. 195:119-129.

I. Yamamoto K., Takahashi T., Asahara T., et al. (2003), “Proliferation,differentiation, and tube formation by endothelial progenitor cells inresponse to shear stress,” J Appl Physiol. 95: 2081-2088.

J. Cohen S., and Leor J. (2004), “Rebuilding Broken Hearts,” ScientificAmerican, November: 45-51.

K. US Patent Application Publication 2004/0228897 to Zhang et al.

L. US Patent Application Publication 2005/0272152 to Xu et al.

SUMMARY OF THE INVENTION

In the context of the present patent application and in the claims, a“core cell population” (CCP) is a population of at least 5 million cellswhich have a density of less than 1.072 g/ml, and at least 1% of whichare CD34+CD45−/Dim (i.e., at least 50,000 of the cells are both (a) CD34positive and (b) CD45 negative or CD45 Dim).

A CCP is typically, but not necessarily, generated from a hematopoieticsource.

For most applications, at least 40% of the CCP is CD31Bright (i.e., atleast 2 million cells out of the 5 million cells are CD31Bright).

While not being limited to any method of detection, cells expressingincreased amounts of CD31 relative to isotype control are termed“CD31Bright” cells, because these cells bear more CD31 moleculesrelative to other cells, and thus tend to fluoresce brightly whenstained with fluorescently-labeled antibodies. In this context, in thespecification and in the claims, “bright” means that the fluorescenceintensity of the labeled cellular marker of interest is at least 50times higher (if measured using flow cytometry) than the isotype controlintensity.

In accordance with an embodiment of the present invention, a method forproducing a progenitor/precursor cell population (PCP) is provided,comprising (a) processing cells extracted from a cell donor to yield aCCP, and (b) stimulating the CCP to differentiate into theprogenitor/precursor cell population. In the context of thespecification and in the claims, “progenitor/precursor” cells arepartially differentiated cells that are able to divide and give rise todifferentiated cells.

While for some applications described herein, the density of the cellsin the CCP is typically less than 1.072 g/ml (as described), for someapplications, the CCP has at least 5 million cells having a density ofless than 1.062 g/ml.

In the context of the specification and in the claims, an “elementalcell population” (ECP) is a population of at least 5 million cells whichhave a density of less than 1.072 g/ml, at least 1.0% of which areCD34+CD45−/Dim, and at least 30% of which are CD31Bright. Typically, butnot necessarily, at least 40% of the cells in the ECP are CD31Bright.Typically, but not necessarily, at least 30% of the cells in the ECP areCD14+. Typically, but not necessarily, at least 1.5% or at least 2% ofthe cells in the ECP are CD34+CD45−/Dim. For some applications, the ECPhas at least 5 million cells having a density of less than 1.062 g/ml.It is typically but not necessarily the case that a CCP is also an ECP.It is noted that, although for simplicity, embodiments of the presentinvention are described herein with respect to procedures relating to aCCP, the scope of the present invention includes, in each instance,performing the same procedure in relation to an ECP.

An “initiating cell population” (ICP), in the context of thespecification and in the claims, is a cell population that candifferentiate into a PCP. CCPs and ECPs are both examples of an ICP. AnICP is typically but not necessarily created by a process that comprisesseparating lower density cells (that are included in the ICP) fromhigher density cells. Such a separation may be accomplished, forexample, by use of one or more gradients.

For some applications, the CCP-derived progenitor cells are used as atherapeutic cell product (e.g., for cancer therapy, for tissueregeneration, for tissue engineering, and/or for tissue replacement), asa research tool (e.g., for research of signal transduction, or forscreening of growth factors), and/or as a diagnostic tool. When theCCP-derived progenitor cells are used as a therapeutic cell product,they are typically administered to a patient, in whom the progenitorcells mature into the desired cells (e.g., endothelial cells, retinalcells, etc.).

In an embodiment, at least one result of at least one stage in a processdescribed herein is used as a diagnostic indicator. For example,pathology of a patient may be indicated if an in vitro procedureperformed on extracted blood of the patient does not produce a CCP, whenthe same procedure performed on cells extracted from a healthy volunteerwould result in production of the CCP. Alternatively or additionally, apathology of a patient may be indicated if an in vitro stimulationprocedure performed on an autologous CCP does not produce a desirednumber of progenitor cells of a particular class, when the sameprocedure would produce the desired number of progenitor cells of aparticular class from a CCP derived from cells of a healthy volunteer.Further alternatively or additionally, a pathology of a patient may beindicated if one or more in vitro protocols used to assess a PCP do notyield the same results as a PCP originated from a healthy volunteer.Still further alternatively or additionally, a pathology of a patientmay be indicated if one or more protocols used to assess a PCP followingimplantation within a patient do not perform as expected (e.g., like aPCP implanted in a healthy animal or human volunteer, or in an animalmodel of a similar disease).

When hematopoietic stem cells are used as a source to create the CCP,the resultant CCP is typically but not necessarily characterized by atleast 40% of the cells in the CCP being CD31Bright, and at least 2.2% orat least 2.5% of the cells being CD34+CD45−/Dim.

Typically, the process of stimulating the CCP takes between about 2 andabout 15 days (e.g., between about 3 and about 15 days), or betweenabout 15 and about 120 days (e.g., between about 15 and about 30 days).Alternatively, stimulating the CCP takes less than 2 days, or more than120 days.

The mammalian cell donor may be human or non-human, as appropriate. Forsome applications, the mammalian cell donor ultimately receives anadministration of a product derived from the CCP, while for otherapplications, the mammalian cell donor does not receive such a product.Stem cells that can be used to produce the CCP are typically but notnecessarily derived from one or more of the following source tissues:embryonic tissue, umbilical cord blood or tissue, neonatal tissue, adulttissue, bone marrow, mobilized blood, peripheral blood, peripheral bloodmononuclear cells, skin cells, and other stem-cell-containing tissue. Itis noted that the stem cells may be obtained from fresh samples of thesesources or from frozen and then thawed cells from these source tissues.

The CCP is typically prepared by generating or obtaining a single cellsuspension from one of the abovementioned source tissues. For example,mobilized blood mononuclear cells may be extracted using a 1.077 g/mldensity gradient, e.g., a Ficoll™ gradient, including copolymers ofsucrose and epichlorohydrin. It is to be noted that such a gradient isnot used for all applications, e.g., for applications in which a singlecell suspension is generated from a non-hematopoietic source (e.g.,mucosal or skin cells). The output of this gradient is then typicallypassed through a second gradient (e.g., a Percoll™ gradient, includingpolyvinylpyrrolidone-coated silica colloids), suitable for selectingcells having a density less than 1.072 g/ml or less than 1.062 g/ml.These selected cells then typically propagate, in vitro, until theybecome a CCP. As appropriate, other density gradients may be used,independently of or in combination with those cited above in order toenrich the designated cells of the CCP. For example, an OptiPrep™gradient, including an aqueous solution of Iodixanol, and/or a Nycodenz™gradient may also be used.

The CCP is typically stimulated to generate progenitor cells of one ormore of the following cell classes:

Blood cells (e.g., red blood cells and/or white blood cells (such as Tcells or B cells));

Neural lineage cells (e.g., CNS neurons, oligodendrocytes, astrocytes,peripheral nervous system (PNS) neurons, and retinal cells (including,but not limited to, photoreceptors, pigment epithelium cells or retinalganglion cells).

Endothelial cells;

Pericytes;

Smooth muscle cells;

Cardiomyocytes;

Osteoblasts;

Pancreatic endocrine or exocrine cells (e.g., beta cells or alphacells);

Hepatic tissue (e.g., hepatocytes); and

Kidney cells.

For some applications, the CCP is transfected with a gene prior to thestimulation of the CCP, whereupon the CCP differentiates into apopulation of desired progenitor cells containing the transfected gene.Typically, these progenitor cells are then administered to a patient.For some applications, the PCP is transfected with a gene. Typically,these PCP cells are then administered to a patient.

In order to stimulate the CCP to differentiate into a desired class ofprogenitor cells, or in association with stimulation of the CCP todifferentiate into a desired class of progenitor cells, the CCP istypically directly or indirectly co-cultured with “target tissue.” The“target tissue” typically but not necessarily includes tissue from anorgan whose cells represent a desired final state of the progenitorcells. For example, the target tissue may include brain or similartissue, or heart or similar tissue, if it is desired for the progenitorcells to differentiate into brain tissue or into heart tissue,respectively. Other examples include:

(a) co-culturing the CCP with peripheral nerves (and/or culturing theCCP in conditioned medium derived therefrom), to induce differentiationof the CCP into peripheral neurons;

(b) co-culturing the CCP with central nervous system (CNS) nerves(and/or culturing the CCP in conditioned medium derived therefrom), toinduce differentiation of the CCP into CNS neurons;

(c) co-culturing the CCP with retinal tissue (and/or culturing the CCPin conditioned medium derived therefrom), to induce differentiation ofthe CCP into retinal tissue. The retinal tissue may include, forexample, one or more of: pigment epithelium, or photoreceptors. Asappropriate, the retinal tissue may comprise fetal retinal tissue,embryonic retinal tissue, or mature retinal tissue;

(d) co-culturing the CCP with blood vessel tissue (and/or culturing theCCP in conditioned medium derived therefrom), to induce differentiationof the CCP into angiogenic lineage tissue and/or cardiomyocytes (CMCs);

(e) co-culturing the CCP with cardiac tissue (and/or culturing the CCPin conditioned medium derived therefrom), to induce differentiation ofthe CCP into CMCs;

(f) co-culturing the CCP with pancreatic endocrine or exocrine tissue(and/or culturing the CCP in conditioned medium derived therefrom), toinduce differentiation of the CCP into pancreatic endocrine or exocrinecells; and

(g) co-culturing the CCP with smooth muscle tissue (and/or culturing theCCP in conditioned medium derived therefrom), to induce differentiationof the CCP into smooth muscle cells.

Techniques described herein with respect to use of a target tissue maybe used with any “sample” tissue, regardless of whether it is desiredfor the CCP to differentiate into a PCP having cells like those in thesample tissue.

For some applications, slices or a homogenate of the target tissue areused for co-culturing, although other techniques for preparing thetarget tissue will be apparent to a person of ordinary skill in the artwho has read the disclosure of the present patent application.

The target tissue may be in essentially direct contact with the CCP, orseparated therefrom by a semi-permeable membrane. As appropriate, thetarget tissue may be autologous, syngeneic, allogeneic, or xenogeneicwith respect to the source tissue from which the CCP was produced.Alternatively or additionally, the CCP is cultured in a conditionedmedium made using target, tissue (e.g., a target tissue describedhereinabove), that is autologous, syngeneic, allogeneic, or xenogeneicwith respect to the source tissue from which the CCP was produced. Forsome applications, the target tissue and the CCP are co-cultured in theconditioned medium. It is to be noted that the source of the targettissue may also be tissue from a cadaver, and/or may be lyophilized,fresh, or frozen.

Alternatively or additionally, for some applications, to produce adesired class of progenitor cells, cells from the CCP are cultured inthe presence of stimulation caused by “stimulation factors,” e.g., oneor more antibodies, cytokines, growth factors, tissue-derived extracellular matrix, and/or other molecules, such as: IL-8, anti-IL-8,anti-CD34, anti-Tie-2, anti-CD133, anti-CD117, LIF, EPO, IGF, b-FGF,M-CSF, GM-CSF, TGF alpha, TGF beta, VEGF, BHA, BDNF, NGF, NT3, NT4/5,GDNF, S-100, CNTF, EGF, NGF3, CFN, ADMIF, estrogen, cortisone,dexamethasone, or any other molecule from the steroid family, prolactin,an adrenocorticoid hormone, ACTH, glutamate, serotonin, acetylcholine,NO, retinoic acid (RA), heparin, insulin, forskolin, a statin, ananti-diabetic drug (e.g., a thiazolidinedione such as rosiglitazone),NO, MCDB-201, MCT-165, glatiramer acetate (L-glutamic acid, L-alanine,L-tyrosine, L-lysine), a glatiramer acetate-like molecule, IFN alpha,IFN beta, or any other immunoregulatory agent, sodium selenite, linoleicacid, ascorbic acid, transferrin, 5-azacytidine, PDGF, VEGF,cardiotrophin, and thrombin.

In the context of the specification and in the claims, a “glatirameracetate-like molecule” means a copolymer comprising:

(a) the same four amino acids as in glatiramer acetate, but in differentratios, (e.g., within 5%, 10%, or 25% of their current values ofL-glutamic acid:L-alanine:L-tyrosine:L-lysine=0.141:0.427:0.095:0.338);

(b) three of the four amino acids in glatiramer acetate, but the fourthamino acid is replaced by a different naturally-occurring or syntheticamino acid;

(c) four amino acids, in which at least one of the amino acids is anenantiomer of the corresponding amino acid in glatiramer acetate, andthe remainder of the amino acids (if any) are the corresponding L-aminoacids that are in glatiramer acetate; or

(d) a combination one or more of (a), (b), and (c).

It is to be appreciated that the particular stimulation factorsdescribed herein are by way of illustration and not limitation, and thescope of the present invention includes the use of other stimulationfactors. As appropriate, these may be utilized in a concentration ofbetween about 100 pg/ml and about 100 μg/ml (or molar equivalents).Typically, particular stimulation factors are selected in accordancewith the particular class of progenitor cells desired. For example, toinduce neural progenitor cells, one or more of the following stimulationfactors are used: BHA, BDNF, NGF, NT3, NT4/5, GDNF, MCT-165, glatirameracetate, a glatiramer acetate-like molecule, IFN alpha, IFN beta or anyother immunoregulatory agent, S-100, CNTF, EGF, NGF3, CFN, ADMIF, andacetylcholine. In another example, to induce CMC progenitors, one ormore of the following stimulation factors are used: bFGF, cortisone,estrogen, progesterone, or any other molecule form the steroid family,NO, sodium selenite, linoleic acid, ascorbic acid, retinoic acid (RA) orany other derivative of vitamin D, transferrin, 5-azacytidine, MCT-165,glatiramer acetate, a glatiramer acetate-like molecule, IFN alpha, IFNbeta, or any other immunoregulatory agent, TGF-beta, insulin, EGF, IGF,PDGF, VEGF, cardiotrophin, MCDB201, and thrombin.

For some applications, the stimulation factors are introduced to the CCPin a soluble form, and/or in an aggregated form, and/or attached to asurface of a culture dish. In an embodiment, the CCP is incubated on asurface comprising a growth-enhancing molecule other than collagen orfibronectin. The growth-enhancing molecule may comprise, for example,VEGF or another suitable antibody or factor described herein. Asappropriate, the growth-enhancing molecule may be mixed with collagen orfibronectin or plasma, or may be coated on the surface in a layerseparate from a layer on the surface that comprises collagen orfibronectin or plasma. Alternatively, the only growth-enhancingmolecule(s) on the surface of the culture dish is collagen and/orfibronectin and/or plasma.

In the context of the present patent application and in the claims, asurface “comprising” or “including” a molecule means that the moleculeis coated on the surface, attached to the surface, or otherwiseintegrated into the surface.

Following stimulation of the CCP, the resultant product is typicallytested to verify that it has differentiated into a desired form.Characterization of the differentiated cells is performed according tothe cells' phenotypical, genotypical and physiological features. Inaccordance with an embodiment of the present invention, the cells arecharacterized by assessing functional/physiological activity thereof, incombination with or in place of evaluating the presence or absence ofcertain cellular markers. Evaluating functional/physiological activityof the cells following the stimulation of the CCP helps increase thelikelihood that the product obtained and designated for in vivo use willperform as expected.

For example, when angiogenic cell precursors (ACPs) (which also includeendothelial progenitor cells (EPCs)) are the desired product, theproduct is typically positive for the generation and/or expression ofone or more of CD34, CD 117, CD133, Tie-2, CD31, CD34+CD133+, KDR,CD34+KDR+, CD144, von Willebrand Factor, SH2 (CD105), SH3, fibronectin,collagen (types I, III and/or IV), ICAM (type 1 or 2), VCAM1, Vimentin,BMP-R IA, BMP-RII, CD44, integrin b1, aSM-actin, and MUC18, CXCR4.Additionally, the ACP product typically functionally demonstrates uptakeof Acetylated-Low Density Lipoprotein (Ac-LDL) (i.e., the product isAc-LDL+) and/or secretes one or more of the following molecules:Interleukin-8 (IL-8), VEGF, Angiogenin, Matrix metalloproteinase 2(MMP-2), or Matrix metalloproteinase 9 (MMP-9). Alternatively oradditionally, the ACP product generates tube-like structures on asemi-solid matrix, and/or migrates towards chemoattractants (such asSDF-1 or VEGF), and/or proliferates in response to cell activation,and/or generates typical cell colonies/clusters. For some applications,in order to further characterize the cells, CD31Bright cells thatdemonstrate uptake of Ac-LDL are examined.

Typically, greater than 1.5% of the core cell population that wasstimulated demonstrates one or more of the abovementionedcharacteristics. Alternatively, if neural progenitor cells are thedesired product, then the product is typically positive for thegeneration and/or the expression of one or more of: Nestin, NSE, Notch,numb, Musashi-1, presenilin, FGFR4, Fz9, SOX 2, CD133, CD15, GD2,rhodopsin, recoverin, calretinin, PAX6, RX, Chx10, O4, and GFAP. Furtheralternatively, if cardiomyocyte (CMC) progenitors are the desiredproduct, then the product is typically positive for the generationand/or the expression of one or more of: CD31, CD117, sarcomericalpha-actin, beta-actin, alpha-actinin, desmin, cardiac troponin T,connexin43, alpha/beta-MHC, sarcomeric alpha-tropomyosin, Troponin I,GATA-4, Nkx2.5/Csx,and MEF-2.

For some applications, the time duration between collecting cells fromthe cell donor and using the CCP-derived progenitor cells (e.g., foradministration into a patient), is reduced in order to effect almostimmediate use thereof. Alternatively, the cells are preserved at one ormore points in the process. For example, the CCP may be frozen prior tothe stimulation thereof that generates progenitor cells. Alternatively,the CCP is stimulated in order to generate desired progenitor cells, andthese progenitor cells are frozen. In either of these cases, the frozencells may be stored and/or transported, for subsequent thawing and use.“Transport,” in the context of the specification and the claims, meanstransport to a remote site, e.g., a site greater than 10 km or 100 kmaway from a site where the CCP is first created.

It is noted that certain applications are suitable for large-scalecommercialization, including freezing and transport, such as (a)generation of stores of CCPs, (b) generation of stores of PCPs, (such ashematopoietic stem cells able to mature into CMCs), and (c) stem cellbanks where individuals may store a CCP or differentiated progenitorcells, for possible later use. Other applications (such as acutepost-stroke autologous administration of neuronal stem cells) may notbenefit, or may not benefit as greatly, from the time delays provided byfreezing of cells, although the technique may be useful for somepurposes.

For some applications, the CCP is cultured for a period lasting betweenabout 1 and about 20 days (e.g., between about 1 and 5 days) in aculture medium comprising less than about 5% serum. Alternatively, theCCP is cultured for a period lasting between about 1 and about 20 days(e.g., between about 1 and about 5 days) in a culture medium comprisinggreater than about 10% serum. In an embodiment, one of these periodsfollows the other of these periods.

For some applications, the CCP is cultured, during a low-serum timeperiod, in a culture medium comprising less than about 10% serum, and,during a high-serum time period, in a culture medium comprising greaterthan or equal to about 10% serum. In an embodiment, culturing the CCPduring the low-serum time period comprises culturing the CCP for aduration of between about 1 and about 20 days (e.g., between about 1 andabout 5 days). Alternatively or additionally, culturing the CCP duringthe high-serum time period comprises culturing the CCP for a duration ofbetween about 1 and about 120 days (e.g., between about 1 and about 30days). Typically, culturing the CCP during the low-serum time period isperformed prior to culturing the CCP during the high-serum time period.Alternatively, culturing the CCP during the low-serum time period isperformed following culturing the CCP during the high-serum time period.

For some applications, during a hypoxic time period lasting at leastabout 2 hours, the CCP is cultured under hypoxic conditions, and, duringa non-hypoxic time period lasting at least about 1 day, the CCP iscultured under non-hypoxic conditions. Culturing the CCP under hypoxicconditions may be performed prior to or following culturing the CCPunder non-hypoxic conditions. Typically, but not necessarily, thehypoxic and non-hypoxic time-periods are within a culturing time periodlasting less than about 120 days (e.g., less than about 30 days), andculturing the CCP under hypoxic conditions comprises culturing the CCPunder hypoxic conditions during the first about two days of theculturing time period. Alternatively or additionally, culturing the CCPunder hypoxic conditions comprises culturing the CCP under hypoxicconditions during the last about two days of the culturing time period.Further alternatively or additionally, culturing the CCP under hypoxicconditions comprises culturing the CCP under hypoxic conditions for atleast about 2 hours between a first two days and a last two days of theculturing time period.

For some applications, the CCP is cultured in a culture mediumcomprising at least one of the following: erythropoietin, a statin, andan antidiabetic agent (e.g., a thiazolidinedione such as rosiglitazone).Alternatively or additionally, the CCP is cultured in the presence ofone or more proliferation-differentiation-enhancing agents, such as,anti-CD34, anti-Tie-2, anti-CD133, anti-CD117, LIF, EPO, IGF, b-FGF,M-CSF, GM-CSF, TGF alpha, TGF beta, VEGF, BHA, BDNF, NGF, NT3, NT4/5,GDNF, S-100, CNTF, EGF, NGF3, CFN, ADMIF, estrogen, prolactin, anadrenocorticoid hormone, ACTH, glutamate, serotonin, acetylcholine, NO,retinoic acid (RA) or any other vitamin D derivative, heparin, insulin,forskolin, cortisone, cortisol, dexamethasone, progesterone, or anyother molecule from the steroid family, a statin, or an anti-diabeticdrug (e.g., a thiazolidinedione such as rosiglitazone), MCDB-201,MCT-165, glatiramer acetate, a glatiramer acetate-like molecule, IFNalpha, IFN beta or any other immunoregulatory agent, sodium selenite,linoleic acid, ascorbic acid, transferrin, 5-azacytidine, PDGF, VEGF,cardiotrophin, and thrombin.

In an embodiment, techniques described herein are practiced incombination with (a) techniques described in one or more of thereferences cited herein, (b) techniques described in U.S. ProvisionalPatent Application 60/576,266, filed Jun. 1, 2004, (c) techniquesdescribed in U.S. Provisional Patent Application 60/588,520, filed Jul.15, 2004, (d) techniques described in U.S. Provisional PatentApplication 60/668,739, filed Apr. 5, 2005, (e) techniques described inU.S. Provisional Patent Application 60/636,391, filed Dec. 14, 2004, (f)techniques described in PCT Patent Application PCT/IL2005/001345, filedDec. 14, 2005, and/or PCT Patent Application PCT/IL2005/001348, filedDec. 14, 2005. Each of these patent applications is assigned to theassignee of the present patent application and is incorporated herein byreference, and the scope of the present invention includes embodimentsdescribed therein.

In an embodiment, a method is provided comprising culturing the CCP in afirst container during a first portion of a culturing period; removingall or at least some cells of the CCP from the first container at theend of the first portion of the period; and culturing, in a secondcontainer during a second portion of the period, the cells removed fromthe first container. For example, removing at least some of the CCPcells may comprise selecting for removal cells that adhere to a surfaceof the first container.

When the cells from a progenitor/precursor cell population (PCP) derivedfrom a CCP are designated for implantation into a human, they should begenerally free from any bacterial or viral contamination. Additionally,in the case of a PCP of angiogenic cell precursors (ACPs), one or moreof the following phenotypical, genotypical and physiological conditionsshould typically be met:

(I) Cells should be morphologically characterized as (a) larger in sizethan 20 uM and/or (b) elongated, spindle-shaped or irregular-shapedand/or (c) granulated or dark nucleated and/or (d) with flagella-likestructures or pseudopodia and/or (e) fibroblast-like or polygonal inshape.

(II) Final cell suspension should typically contain at least 1 millioncells expressing one or more of the following markers: CD31Bright, CD34,CD117, CD133, Tie-2, CD34+CD133+, KDR, CD34+KDR+, CD144, von WillebrandFactor, SH2 (CD105), SH3, fibronectin, collagen (types I, III and/orIV), ICAM (type 1 or 2), VCAM1, Vimentin, BMP-R IA, BMP-RII, CD44,integrin b1, aSM-actin, and MUC18, CXCR4

(III) Cells should be able to uptake Ac-LDL.

(IV) Cells expressing CD31Bright should also demonstrate the ability touptake Ac-LDL (e.g., at least about 10% or about 25% of cells that areCD31Bright also are able to uptake Ac-LDL).

(V) Cells should generally secrete one or more of the followingmolecules: IL-8, Angiogenin, VEGF, MMP2, and MMP9.

(VI) Cells should generally form tube-like structures when cultured on asemi-solid matrix containing growth factors.

(VII) Cells should generally migrate chemotactically towards differentchemoattractants, such as SDF-1 and VEGF.

(VIII) Cells should generally form typical colonies and/or clusters whencultured in medium supplemented with growth factors such as VEGF andGM-SCF.

It is noted that the cells in CCPs generated from various tissuestypically can be characterized as having greater than 75% viability.

It is noted that CCPs generated from blood, bone marrow, and umbilicalcord blood, typically have greater than 70% of their cells being CD45+.

In some embodiments of the present invention, a novel composition ofmatter is provided, comprising (a) a cell population, or (b) a mixturecomprising a cell population and molecules produced by the cellpopulation, wherein (a) or (b) are produced by a method described herein(for example, in one of the methods set forth in the followingparagraphs preceding the Brief Description section of the present patentapplication, or in one of the methods described in the DetailedDescription section of the present patent application).

There is therefore provided, in accordance with an embodiment of theinvention, a composition of matter, including a population of culturedcells that includes a sub-population of cells that both stain asCD31Bright and demonstrate uptake of Ac-LDL+.

In an embodiment, the sub-population includes at least 10%, 25%, or 50%of the cells in the population.

In an embodiment, at least 1.5% of the cells of the population includeat least one morphological feature selected from the group consistingof: a cell size larger than 20 um, an elongated cell, a spindle-shapedcell, an irregularly-shaped cell, a granulated cell, a cell including anenlarged dark nucleus, a multinuclear cell, a cell includingflagella-like structures, a cell including pseudopodia, and a cellhaving a polygonal shape.

In an embodiment, at least 1.5% of the cells of the population includeat least one feature selected from the group consisting of: CD34, CD117,CD133, Tie-2, CD34+CD133+, KDR, CD34+KDR+, CD144, von Willebrand Factor,SH2 (CD105), SH3, fibronectin, collagen type I, collagen type III,collagen type IV, ICAM type 1, ICAM type 2, VCAM1, vimentin, BMP-R IA,BMP-RII, CD44, integrin b1, aSM-actin, MUC18, and CXCR4.

In an embodiment, at least 1.5% of the cells of the population secreteat least one molecule selected from the group consisting of: IL-8,angiogenin, VEGF, MMP2, and MMP9.

In an embodiment, at least 1.5% of the cells of the population includeat least one feature selected from the group consisting of: a tube-likestructure, a tendency to form a colony, a tendency to form a cluster,and a tendency to migrate towards a chemoattractant.

There is further provided, in accordance with an embodiment of theinvention, a method including in vitro stimulating an initiating cellpopulation (ICP) of at least 5 million cells that have a density of lessthan 1.072 g/ml, at least 1% of which are CD34+CD45−/Dim, and at least25% of which are CD31Bright, to differentiate into aprogenitor/precursor cell population (PCP).

There is still further provided, in accordance with an embodiment of theinvention, a method including in vitro stimulating an initiating cellpopulation (ICP) of at least ten thousand cells that have a density ofless than 1.072 g/ml and at least 25% of which are CD31Bright todifferentiate into a progenitor/precursor cell population (PCP).

There is yet further provided, in accordance with an embodiment of theinvention, a method including separating lower density cells from higherdensity cells, the lower density cells defining an initiating cellpopulation (ICP) at least 40% of which are CD31Bright, and in vitrostimulating the ICP to differentiate into a progenitor/precursor cellpopulation (PCP).

In an embodiment, stimulating the ICP includes culturing the ICP for aperiod lasting between 1 and 5 days in a culture medium including lessthan or equal to 10% serum.

In an embodiment, stimulating the ICP includes culturing the ICP for aperiod lasting between 1 and 5 days in a culture medium including lessthan or equal to 5% serum.

In an embodiment, stimulating the ICP includes culturing the ICP for aperiod lasting between 1 and 5 days in a culture medium including 5-10%serum.

In an embodiment, stimulating the ICP includes culturing the ICP for aperiod lasting between 1 and 5 days in a culture medium including lessthan or equal to 5% serum.

In an embodiment, stimulating the ICP includes culturing the ICP for aperiod lasting between 1 and 5 days in a culture medium including atleast 10% serum.

In an embodiment, stimulating the ICP includes culturing the ICP in aculture medium including a factor selected from the group consisting of:anti-Tie-2, anti-CD133, and anti-CD 117.

In an embodiment, stimulating the ICP includes culturing the ICP in aculture medium including a factor selected from the group consisting of:anti-Tie-2, anti-CD133, and anti-CD117, anti-IL-8, anti IL-8 receptor,IL-8-antagonist, VEGF, anti-VEGF, and anti-VEGF receptor.

In an embodiment, stimulating the ICP includes culturing the ICP in aculture medium including IL-8.

In an embodiment, stimulating the ICP includes culturing the ICP in thepresence of a factor selected from the group consisting of: anti IL-8receptor, IL-8-antagonist, VEGF, anti-VEGF, and anti-VEGF receptor.

In an embodiment, stimulating the ICP includes culturing the ICP in thepresence of IL-8.

In an embodiment, characterizing the PCP includes characterizing the PCPin response to an identification in the PCP of CXCR8.

In an embodiment, characterizing the PCP includes identifying that atleast 1.5% of cells of the PCP include CXCR8.

In an embodiment, characterizing the PCP includes culturing a portion ofthe PCP on a semi-solid extracellular matrix (ECM), and identifying inthe cultured portion a feature selected from the group consisting of: atube-like structure, a colony, a cluster, and a tendency to migratetowards a chemoattractant.

In an embodiment, characterizing the PCP includes culturing at least aportion of the PCP on a membrane, and identifying a tendency of the atleast a portion of the PCP to migrate toward IL-8.

In an embodiment, the ICP includes at least 5 million cells, andstimulating the ICP includes stimulating the ICP that includes the atleast 5 million cells.

In an embodiment, at least 1.5% of the cells of the ICP areCD34+CD45−/Dim, and stimulating the ICP includes stimulating the ICP ofwhich at least 1.5% of the cells are CD34+CD45−/Dim.

In an embodiment, at least 2% of the cells of the ICP areCD34+CD45−/Dim, and stimulating the ICP includes stimulating the ICP ofwhich at least 2% of the cells are CD34+CD45−/Dim.

In an embodiment, at least 30% of the cells of the ICP are CD31Bright,and stimulating the ICP includes stimulating the ICP of which at least30% of the cells are CD31Bright.

In an embodiment, the ICP includes at least 5 million cells that have adensity of less than 1.062 g/ml, at least 1% of which areCD34+CD45−/Dim, and stimulating the ICP includes stimulating the ICPthat has the at least 5 million cells that have a density of less than1.062 g/ml.

In an embodiment, at least 50% of cells in the ICP are CD31Bright, andstimulating the ICP includes stimulating the ICP of which at least 50%of cells therein are CD31Bright.

In an embodiment, the method includes preparing the PCP as a product foradministration to a patient. Alternatively or additionally, the methodincludes preparing the PCP as a research tool.

In an embodiment, stimulating the ICP includes only stimulating the ICPif the ICP is derived from a mammalian donor.

In an embodiment, the method includes applying cells extracted from amammalian donor to one or more gradients suitable for selecting cellshaving a density less than 1.072 g/ml, and deriving the ICP from thecells applied to the gradient.

In an embodiment, the ICP is characterized by at least 2.5% of the ICPbeing CD34+CD45−/Dim, and stimulating the ICP includes stimulating theICP having the at least 2.5% of the ICP that are CD34+CD45−/Dim.

In an embodiment, the ICP is characterized by at least 40% of the ICPbeing CD31Bright, and stimulating the ICP includes stimulating the ICPhaving the at least 40% of the ICP that are CD31Bright.

In an embodiment, stimulating the ICP includes stimulating the ICP todifferentiate into a pre-designated, desired class of progenitor cells.

In an embodiment, the method includes deriving the ICP from at least onesource selected from the group consisting of embryonic tissue, fetaltissue, umbilical cord blood, umbilical cord tissue, neonatal tissue,adult tissue, bone marrow, mobilized blood, peripheral blood, peripheralblood mononuclear cells, skin cells, and plant tissue.

In an embodiment, the method includes deriving the ICP from at least onesource selected from the group consisting of: fresh tissue and frozentissue.

In an embodiment, the method includes identifying an intended recipientof the PCP, and deriving the ICP from at least one source selected fromthe group consisting of tissue autologous to tissue of the intendedrecipient, tissue syngeneic to tissue of the intended recipient, tissueallogeneic to tissue of the intended recipient, and tissue xenogeneic totissue of the intended recipient.

In an embodiment, stimulating the ICP includes culturing the ICP for aperiod lasting between 1 and 5 days in a culture medium including lessthan 5% serum.

In an embodiment, stimulating the ICP includes culturing the ICP for aperiod lasting between 1 and 5 days in a culture medium including atleast 10% serum.

In an embodiment, stimulating the ICP includes culturing the ICP in aculture medium including a factor selected from the group consisting of:erythropoietin, a statin, and an antidiabetic agent.

In an embodiment, stimulating the ICP includes culturing the ICP in aculture medium including a factor selected from the group consisting of:estrogen, prolactin, progestin, an adrenocorticoid hormone, ACTH, andcortisone.

In an embodiment, stimulating the ICP includes culturing the ICP in aculture medium including a factor selected from the group consisting of:anti-Tie-2, anti-CD133, and anti-CD117.

In an embodiment, stimulating the ICP includes culturing the ICP in thepresence of a factor selected from the group consisting of:erythropoietin, a statin, an antidiabetic agent, a thiazolidinedione,rosiglitazone, a proliferation-differentiation-enhancing agent,anti-CD34, anti-Tie-2, anti-CD133, anti-CD117, LIF, EPO, IGF, b-FGF,M-CSF, GM-CSF, TGF alpha, TGF beta, VEGF, BHA, BDNF, GDNF, NGF, NT3,NT4/5, S-100, CNTF, EGF, NGF3, CFN, ADMIF, estrogen, prolactin, anadrenocorticoid hormone, ACTH, MCT-165, glatiramer acetate, a glatirameracetate-like molecule, IFN alpha, IFN beta, glutamate, serotonin,acetylcholine, NO, retinoic acid (RA), heparin, insulin, cortisone, andforskolin.

In an embodiment, the method includes preparing the ICP, andfacilitating a diagnosis responsive to a characteristic of thepreparation of the ICP.

In an embodiment, the method includes freezing the ICP prior tostimulating the ICP.

In an embodiment, the method includes freezing the PCP.

In an embodiment, the method includes transporting the ICP to a site atleast 10 km from a site where the ICP is first created, and stimulatingthe ICP at the remote site.

In an embodiment, the method includes transporting the PCP to a site atleast 10 km from a site where the PCP is first created.

In an embodiment, the method includes identifying the PCP as beingsuitable for therapeutic implantation in response to an assessment thatthe PCP includes at least 1 million PCP cells.

In an embodiment, the method includes identifying the PCP as beingsuitable for therapeutic implantation in response to an assessment thatat least 1.5% of cells of the PCP demonstrate a feature selected fromthe group consisting of: a desired morphology, a desired cellularmarker, a desired cellular component, a desired enzyme, a desiredreceptor, a desired genotypic feature, and a desired physiologicalfeature.

In an embodiment, the method includes identifying the PCP as beingsuitable for therapeutic implantation in response to an assessment thatthe PCP includes at least 1 million angiogenic cell precursors (ACPs).

In an embodiment, the method includes identifying the PCP as beingsuitable for therapeutic implantation in response to an assessment thatthe PCP includes at least 1 million cardiomyocyte progenitors.

In an embodiment, the method includes identifying the PCP as beingsuitable for therapeutic implantation in response to an assessment thatthe PCP includes at least 1 million neural cell progenitors.

In an embodiment, the method includes transfecting into the PCP a geneidentified as suitable for gene therapy.

In an embodiment, the method includes transfecting a gene into the PCP,and subsequently assessing a level of expression of the gene.

In an embodiment, the method includes transfecting a gene into the ICP,and subsequently assessing a level of expression of the gene.

In an embodiment, stimulating the ICP includes culturing the ICP duringa period of between 2 and 120 days.

In an embodiment, stimulating the ICP includes culturing the ICP duringa period of between 3 and 60 days.

In an embodiment, stimulating the ICP includes culturing the ICP in aculture medium including less than 10% serum, for a duration of between1 and 120 days.

In an embodiment, stimulating the ICP includes culturing the ICP in aculture medium including at least 10% serum, for a duration of between 1and 120 days.

In an embodiment, the method includes characterizing the PCP asincluding angiogenic cell precursors (ACPs), in response to anevaluation of at least a feature selected from the group consisting of:a phenotypical feature of cells in the PCP, a genotypical feature ofcells in the PCP, and a physiological feature of cells in the PCP.

In an embodiment, characterizing the PCP includes characterizing the PCPin response to an evaluation of at least two of the features.

In an embodiment, characterizing the PCP includes characterizing the PCPin response to an evaluation of each of the features.

In an embodiment:

the phenotypical feature includes a morphological feature selected fromthe group consisting of: a cell size larger than 20 μm, an elongatedcell, a spindle-shaped cell, an irregularly-shaped cell, a granulatedcell, a cell including an enlarged dark nucleus, a multinuclear cell, acell including flagella-like structures, a cell including pseudopodia,and a cell having a polygonal shape; and

characterizing the PCP includes characterizing the PCP in response to anevaluation of the selected morphological feature.

In an embodiment, characterizing the PCP includes identifying that atleast 1.5% of cells of the PCP have the selected feature.

In an embodiment, characterizing the PCP includes characterizing the PCPin response to an identification in the PCP of a feature selected fromthe group consisting of: CD31, CD31Bright, CD34, CD117, CD133, Tie-2,CD34+CD133+, KDR, CD34+KDR+, CD144, von Willebrand Factor, SH2 (CD105),SH3, fibronectin, collagen type I, collagen type III, collagen type IV,ICAM type 1, ICAM type 2, VCAM1, vimentin, BMP-R IA, BMP-RII, CD44,integrin b1, aSM-actin, MUC18, and CXCR4.

In an embodiment, characterizing the PCP includes identifying that atleast 1.5% of cells of the PCP have the selected feature.

In an embodiment, characterizing the PCP includes characterizing the PCPin response to an assessment of uptake by the PCP of Ac-LDL.

In an embodiment, characterizing the PCP includes identifying that atleast 1.5% of cells of the PCP demonstrate uptake of Ac-LDL.

In an embodiment, the PCP includes CD31Bright PCP cells, andcharacterizing the PCP includes identifying that at least 10% of theCD31Bright PCP cells demonstrate uptake of Ac-LDL.

In an embodiment, characterizing the PCP includes assessing secretion bythe PCP of a molecule selected from the group consisting of: IL-8,angiogenin, VEGF, MMP2, and MMP9.

In an embodiment, characterizing the PCP includes identifying that atleast 1.5% of cells of the PCP secrete the selected molecule.

In an embodiment, characterizing the PCP includes culturing a portion ofthe PCP on a semi-solid extracellular matrix (ECM), and identifying inthe cultured portion a feature selected from the group consisting of: atube-like structure, a colony, a cluster, and a tendency to migratetowards a chemoattractant.

In an embodiment, characterizing the PCP includes identifying that atleast 1.5% of cells in the cultured portion have a property selectedfrom the group consisting of: formation of a tube-like structure, anability to form a colony, a cluster, and a tendency to migrate towards achemoattractant.

In an embodiment, the method includes identifying the PCP as beingsuitable for therapeutic implantation in response to an assessment thatthe PCP includes at least 1 million ACPs.

In an embodiment, the method includes characterizing the PCP asincluding a cardiomyocyte (CMC) PCP in response to an evaluation of afeature selected from the group consisting of: a phenotypic feature ofcells in the PCP, a genotypic feature on the cells in the PCP, and aphysiological feature of cells in the PCP.

In an embodiment, characterizing the PCP includes characterizing the PCPin response to an evaluation of at least two of the features.

In an embodiment, characterizing the PCP includes characterizing the PCPin response to an evaluation of each of the features.

In an embodiment, the phenotypic feature includes a morphologicalfeature selected from the group consisting of: a cell size larger than20 um, an elongated cell, an irregularly-shaped cell, a granulated cell,a cell including an enlarged dark nucleus, a multinuclear cell, a cellwith dark cytoplasm, and cells arranged in parallel to each other; and

characterizing the PCP includes characterizing the PCP in response to anevaluation of the selected morphological feature.

In an embodiment, characterizing the PCP includes characterizing the PCPin response to an identification in the PCP of a feature selected fromthe group consisting of: CD31, CD117, sarcomeric alpha-actin,beta-actin, alpha-actinin, desmin, cardiac troponin T, Connexin-43,alpha/beta-MHC, sarcomeric alpha-tropomyosin, Troponin I, GATA-4,Nkx2.5/Csx, MLC-2, and MEF-2.

In an embodiment, characterizing the PCP includes characterizing the PCPin response to an identification of the PCP. as expressing a gene for afactor selected from the group consisting of: sarcomeric alpha-actin,beta-actin, alpha-actinin, desmin, cardiac troponin T, Connexin-43,alpha/beta-MHC, sarcomeric alpha-tropomyosin, Troponin I, GATA-4,Nkx2.5/Csx, MLC-2 and MEF-2.

In an embodiment, the method includes identifying the PCP as beingsuitable for therapeutic implantation in response to an assessment thatthe PCP includes at least 1 million CMC progenitors.

In an embodiment, characterizing the PCP includes identifying that atleast 1.5% of cells of the PCP have a characteristic selected from thegroup consisting of a CMC-progenitor morphological characteristic,expression of a CMC-associated cellular marker, expression of aCMC-progenitor gene product, and expression of a CMC-progenitorphysiological feature.

In an embodiment, characterizing the PCP includes characterizing the PCPin response to an identification in the PCP of an action in response toactivation of the PCP, the action selected from the group consisting of:increasing intracellular Ca2⁺ level, generating membranalelectrophysiological action potentials, and mechanical cellularcontraction in vitro.

In an embodiment, activating the PCP to produce the selected action,using a technique selected from the group consisting of: electricalactivation of the PCP, and chemical activation of the PCP.

In an embodiment, the method includes:

assessing a phenotypic aspect of the PCP and a genotypic aspect of thePCP and a physiological aspect of the PCP; and

designating the PCP as being suitable for implantation in a patient inresponse to each of the assessments.

In an embodiment, assessing the phenotypic aspect of the PCP includesassessing an aspect of the PCP selected from the group consisting of:morphology of the PCP, a cellular marker of cells of the PCP, an enzymeof the PCP, a coenzyme of the PCP, and presence of a designated cellularreceptor on cells of the PCP.

In an embodiment, assessing the genotypic aspect of the PCP includesassessing an aspect of the PCP selected from the group consisting of:production of a gene by cells of the PCP, expression of a gene by cellsof the PCP, and generation of a gene product by cells of the PCP.

In an embodiment, assessing the physiological aspect of the PCP includesassessing an aspect of the PCP selected from the group consisting of:secretion of soluble molecules by cells of the PCP, generation ofsignals by cells of the PCP, response by cells of the PCP to signals,and an enzymatic reaction performed by cells of the PCP.

In an embodiment, the method includes facilitating a diagnosisresponsive to stimulating the ICP to differentiate into the PCP.

In an embodiment, facilitating the diagnosis includes assessing anextent to which the stimulation of the TCP produces a particularcharacteristic of the PCP.

In an embodiment, the method includes transfecting a gene into the ICPprior to stimulating the ICP.

In an embodiment, transfecting the gene includes transfecting into theICP a gene identified as suitable for gene therapy.

In an embodiment, the method includes preparing, as a product foradministration to a patient, the PCP generated by differentiation of theICP into which the gene has been transfected.

In an embodiment, stimulating the ICP includes incubating the ICP in acontainer having a surface including a growth-enhancing factor.

In an embodiment, the growth-enhancing factor is selected from the groupconsisting of: collagen, plasma, fibronectin, a growth factor,tissue-derived extra cellular matrix, and an antibody to a stem cellsurface receptor.

In an embodiment, stimulating the ICP includes incubating the TCP in acontainer with a surface including a growth-enhancing molecule otherthan collagen or fibronectin.

In an embodiment, incubating the ICP includes incubating the ICP in acontainer having a surface that includes, in addition to thegrowth-enhancing molecule, at least one of: collagen and fibronectin.

In an embodiment, the method includes mixing the growth-enhancingmolecule with the at least one of: collagen and fibronectin.

In an embodiment, the method includes applying to the surface a layerthat includes the growth-enhancing molecule and a separate layer thatincludes the at least one of: collagen and fibronectin.

In an embodiment, stimulating the ICP includes:

during a low-serum time period, culturing the ICP in a culture mediumincluding less than 10% serum; and

during a high-serum time period, culturing the ICP in a culture mediumincluding greater than or equal to 10% serum.

In an embodiment, culturing the ICP during the low-serum time periodincludes culturing the ICP for a duration of between 1 and 60 days.

In an embodiment, culturing the ICP during the low-serum time periodincludes culturing the ICP for a duration of between 1 and 5 days.

In an embodiment, culturing the ICP during the high-serum time periodincludes culturing the ICP for a duration of between 1 and 120 days.

In an embodiment, culturing the ICP during the high-serum time periodincludes culturing the ICP for a duration of between 1 and 60 days.

In an embodiment, culturing the ICP during the low-serum time period isperformed prior to culturing the ICP during the high-serum time period.

In an embodiment, culturing the ICP during the low-serum time period isperformed following culturing the ICP during the high-serum time period.

In an embodiment, the method includes:

during a hypoxic time period lasting at least 2 hours, culturing the ICPunder hypoxic conditions; and

during a non-hypoxic time period lasting at least 1 day, culturing theICP under non-hypoxic conditions.

In an embodiment, the hypoxic and non-hypoxic time-periods are within aculturing time period lasting less than 30 days, and culturing the ICPunder hypoxic conditions includes culturing the cells under hypoxicconditions during a first two days of the culturing time period.

In an embodiment, the hypoxic and non-hypoxic time-periods are within aculturing time period lasting less than 30 days, and culturing the ICPunder hypoxic conditions includes culturing the ICP under hypoxicconditions during a last two days of the culturing time period.

In an embodiment, the hypoxic and non-hypoxic time-periods are within aculturing time period lasting less than 30 days, and culturing the ICPunder hypoxic conditions includes culturing the ICP under hypoxicconditions for at least 2 hours between a first two days and a last twodays of the culturing time period.

In an embodiment, culturing the ICP under hypoxic conditions isperformed prior to culturing the ICP under non-hypoxic conditions.

In an embodiment, culturing the ICP under hypoxic conditions isperformed following culturing the ICP under non-hypoxic conditions.

In an embodiment, stimulating the ICP includes:

culturing the ICP in a first container during a first portion of aculturing period;

removing at least some cells of the ICP from the first container at theend of the first portion of the period; and

culturing, in a second container during a second portion of the period,the cells removed from the first container.

In an embodiment, the method includes subsequently to the step ofculturing in the second container:

culturing the ICP in a primary container during a first portion of anadditional culturing period;

removing at least some cells of the ICP from the primary container atthe end of the first portion of the additional period; and

culturing, in a secondary container during a second portion of theadditional period, the cells removed from the primary container.

In an embodiment, stimulating the ICP includes:

culturing the ICP in a first container during a first portion of aculturing period;

removing cells of the ICP from the first container at the end of thefirst portion of the period; and

culturing, in a second container during a second portion of the period,the cells removed from the first container.

In an embodiment, removing at least some cells of the ICP includesselecting for removal cells that adhere to a surface of the firstcontainer.

In an embodiment, removing at least some cells of the ICP includesselecting for removal cells that do not adhere to a surface of the firstcontainer.

In an embodiment, the first container includes on a surface thereof agrowth-enhancing molecule, and culturing the ICP in the first containerincludes culturing the ICP in the first container that includes thegrowth-enhancing molecule.

In an embodiment, the growth-enhancing molecule is selected from thegroup consisting of: collagen, plasma, fibronectin, a growth factor,tissue-derived extra cellular matrix and an antibody to a stem cellsurface receptor.

In an embodiment, the second container includes on a surface thereof agrowth-enhancing molecule, and culturing the ICP in the second containerincludes culturing the ICP in the second container that includes thegrowth-enhancing molecule.

In an embodiment, the growth-enhancing molecule is selected from thegroup consisting of: collagen, fibronectin, a growth factor, and anantibody to a stem cell surface receptor.

In an embodiment, stimulating includes culturing the ICP with at leastone factor derived from a sample tissue.

In an embodiment, the method includes preparing a conditioned medium forculturing the ICP therein, the conditioned medium including the factor,the factor being derived from the tissue, the tissue being selected fromthe group consisting of: peripheral nerve tissue, central nervous system(CNS) tissue, retinal tissue, pigment epithelial tissue, photoreceptortissue, fetal retinal tissue, embryonic retinal tissue, mature retinaltissue, blood vessel tissue, cardiac tissue, pancreatic endocrinetissue, pancreatic exocrine tissue, smooth muscle tissue, lymphatictissue, hepatic tissue, lung tissue, skin tissue, exocrine glandulartissue, mammary gland tissue, endocrine glandular tissue, thyroid glandtissue, pituitary gland tissue, and plant tissue.

In an embodiment, stimulating includes co-culturing the ICP with asample tissue.

In an embodiment, co-culturing includes preparing the sample tissue by amethod selected from the group consisting of slicing the sample tissue,and homogenizing the sample issue.

In an embodiment, co-culturing includes: utilizing the sample tissue toproduce a conditioned medium; and co-culturing the ICP with the sampletissue in the conditioned medium.

In an embodiment, co-culturing includes separating the sample tissuefrom the ICP by a semi-permeable membrane.

In an embodiment, designating the sample tissue to include a tissueselected from the group consisting of: peripheral nerve tissue, centralnervous system (CNS) tissue, retinal tissue, pigment epithelial tissue,photoreceptor tissue, fetal retinal tissue, embryonic retinal tissue,mature retinal tissue, blood vessel tissue, cardiac tissue, pancreaticendocrine tissue, pancreatic exocrine tissue, smooth muscle tissue,lymphatic tissue, hepatic tissue, lung tissue, skin tissue, exocrineglandular tissue, mammary gland tissue, endocrine glandular tissue,thyroid gland tissue, pituitary gland tissue, and plant tissue.

In an embodiment, the method includes systemically administering the PCPto a patient.

In an embodiment, the method includes locally administering the PCP to asite of the patient including injured tissue.

In an embodiment, locally administering the PCP includes implanting atthe site a device including the PCP.

In an embodiment, the device includes at least one item selected fromthe group consisting of a metal, a plastic, a glass, and a biodegradableelement, and implanting the device includes implanting the deviceincluding the selected item.

In an embodiment, the method includes using the device to enableincreased survival of PCP in injured tissue

In an embodiment, the method includes configuring the device for slowrelease of cells of the PCP into the injured tissue.

In an embodiment, the method includes secreting, from the PCP,therapeutic molecules to the tissue.

In an embodiment, the method includes secreting, from the device,soluble molecules that support the PCP.

There is also provided, in accordance with an embodiment of theinvention, apparatus for implantation in a patient, including a medicaldevice including a PCP produced according to any of the proceduresdescribed herein for producing a PCP.

In an embodiment, the medical device includes a chamber having the PCPdisposed therein, and a membrane, through which molecules generated bythe PCP are able to pass.

There is further provided, in accordance with an embodiment of theinvention, a method including in vitro stimulating an initiating cellpopulation (ICP) of at least 5 million cells that have a density of lessthan 1.072 g/ml, and at least 1% of which are CD34+CD45−/Dim, todifferentiate into a progenitor/precursor cell population (PCP).

There is also provided, in accordance with an embodiment of theinvention, a method including in vitro stimulating an initiating cellpopulation (ICP) of at least ten thousand cells that have a density ofless than 1.072 g/ml to differentiate into a progenitor/precursor cellpopulation (PCP).

There is further provided, in accordance with an embodiment of theinvention, a method including separating lower density cells from higherdensity cells, the lower density cells defining an initiating cellpopulation (ICP), and in vitro stimulating the ICP to differentiate intoa progenitor/precursor cell population (PCP).

In an embodiment, the ICP includes at least 5 million cells, and whereinstimulating the ICP includes stimulating the ICP that includes the atleast 5 million cells.

In an embodiment, at least 1.5% of the cells of the ICP areCD34+CD45−/Dim, and wherein stimulating the ICP includes stimulating theICP of which at least 1.5% of the cells are CD34+CD45−/Dim.

In an embodiment, at least 2% of the cells of the ICP areCD34+CD45−/Dim, and wherein stimulating the ICP includes stimulating theICP of which at least 2% of the cells are CD34+CD45−/Dim.

In an embodiment, at least 30% of the cells of the ICP are CD31Bright,and wherein stimulating the ICP includes stimulating the ICP of which atleast 30% of the cells are CD34+CD45−/Dim.

In an embodiment, the ICP includes at least 5 million cells that have adensity of less than 1.062 g/ml, at least 1% of which areCD34+CD45−/Dim, and wherein stimulating the ICP includes stimulating theICP that has the at least 5 million cells that have a density of lessthan 1.062 g/ml.

In an embodiment, at least 50% of cells in the ICP are CD31Bright, andwherein stimulating the ICP includes stimulating the ICP of which atleast 50% of cells therein are CD31Bright.

In an embodiment, the method includes preparing the PCP as a product foradministration to a patient.

In an embodiment, the method includes preparing the PCP as a researchtool.

In an embodiment, stimulating the ICP includes only stimulating the ICPif the ICP is derived from a mammalian donor.

In an embodiment, the method includes applying cells extracted from amammalian donor to one or more gradients suitable for selecting cellshaving a density less than 1.072 g/ml, and deriving the ICP from thecells applied to the gradient.

In an embodiment, the ICP is characterized by at least 2.5% of the ICPbeing CD34+CD45−/Dim, and wherein stimulating the ICP includesstimulating the ICP having the at least 2.5% of the ICP that areCD34+CD45−/Dim.

In an embodiment, the ICP is characterized by at least 50% of the ICPbeing CD31Bright, and wherein stimulating the ICP includes stimulatingthe ICP having the at least 50% of the ICP that are CD31Bright.

In an embodiment, the ICP is characterized by at least 40% of the ICPbeing CD31Bright, and wherein stimulating the ICP includes stimulatingthe ICP having the at least 40% of the ICP that are CD31Bright.

In an embodiment, stimulating the ICP includes stimulating the ICP todifferentiate into a pre-designated, desired class of progenitor cells.

In an embodiment, the method includes deriving the ICP from at least onesource selected from the group consisting of: embryonic tissue, fetaltissue, umbilical cord blood, umbilical cord tissue, neonatal tissue,adult tissue, bone marrow, mobilized blood, peripheral blood, peripheralblood mononuclear cells, skin cells, and plant tissue.

In an embodiment, the method includes deriving the ICP from at least onesource selected from the group consisting of: fresh tissue and frozentissue.

In an embodiment, the method includes identifying an intended recipientof the PCP, and deriving the ICP from at least one source selected fromthe group consisting of: tissue autologous to tissue of the intendedrecipient, tissue syngeneic to tissue of the intended recipient, tissueallogeneic to tissue of the intended recipient, and tissue xenogeneic totissue of the intended recipient.

In an embodiment, stimulating the ICP includes culturing the ICP for aperiod lasting between 1 and 5 days in a culture medium including lessthan 5% serum.

In an embodiment, stimulating the ICP includes culturing the ICP for aperiod lasting between 1 and 5 days in a culture medium including atleast 10% serum.

In an embodiment, stimulating the ICP includes culturing the ICP in aculture medium including a factor selected from the group consisting of:erythropoietin, a statin, and an antidiabetic agent.

In an embodiment, stimulating the ICP includes culturing the ICP in aculture medium including a factor selected from the group consisting of:estrogen, prolactin, progestin, an adrenocorticoid hormone, ACTH, andcortisone.

In an embodiment, stimulating the ICP includes culturing the ICP in aculture medium including a factor selected from the group consisting of:anti-Tie-2, anti-CD133, and anti-CD 117.

In an embodiment, stimulating the ICP includes culturing the ICP in thepresence of a factor selected from the group consisting of:erythropoietin, a statin, an antidiabetic agent, a thiazolidinedione,rosiglitazone, a proliferation-differentiation-enhancing agent,anti-CD34, anti-Tie-2, anti-CD133, anti-CD117, LIF, EPO, IGF, b-FGF,M-CSF, GM-CSF, TGF alpha, TGF beta, VEGF, BHA, BDNF, GDNF, NGF, NT3,NT4/5, S-100, CNTF, EGF, NGF3, CFN, ADMIF, estrogen, prolactin, anadrenocorticoid hormone, ACTH, MCT-165, glatiramer acetate, a glatirameracetate-like molecule, IFN alpha, IFN beta or any other immunoregulatoryagent, glutamate, serotonin, acetylcholine, NO, retinoic acid (RA) orany other vitamin D derivative, heparin, insulin, and forskolin,cortisone.

In an embodiment, the method includes preparing the ICP, andfacilitating a diagnosis responsive to a characteristic of thepreparation of the ICP.

In an embodiment, the method includes freezing the ICP prior tostimulating the ICP.

In an embodiment, the method includes freezing the PCP.

In an embodiment, the method includes transporting the ICP to a site atleast 10 km from a site where the ICP is first created, and stimulatingthe ICP at the remote site.

In an embodiment, the method includes transporting the PCP to a site atleast 10 km from a site where the PCP is first created.

In an embodiment, the method includes identifying the PCP as beingsuitable for therapeutic implantation in response to an assessment thatthe PCP includes at least 1 million PCP cells.

In an embodiment, the method includes identifying the PCP as beingsuitable for therapeutic implantation in response to an assessment thatat least 1.5% of cells of the PCP demonstrate a feature selected fromthe group consisting of: a desired morphology, a desired cellularmarker, a desired cellular component, a desired enzyme, a desiredreceptor, a desired genotypic feature, and a desired physiologicalfeature.

In an embodiment, the method includes identifying the PCP as beingsuitable for therapeutic implantation in response to an assessment thatthe PCP includes at least 1 million angiogenic cell precursors (ACPs).

In an embodiment, the method includes identifying the PCP as beingsuitable for therapeutic implantation in response to an assessment thatthe PCP includes at least 1 million cardiomyocyte progenitors.

In an embodiment, the method includes identifying the PCP as beingsuitable for therapeutic implantation in response to an assessment thatthe PCP includes at least 1 million neural cell progenitors.

In an embodiment, the method includes transfecting into the PCP a geneidentified as suitable for gene therapy.

In an embodiment, the method includes transfecting a gene into the PCP,and subsequently assessing a level of expression of the gene.

In an embodiment, the method includes transfecting a gene into the ICP,and subsequently assessing a level of expression of the gene.

In an embodiment, stimulating the ICP includes culturing the ICP duringa period of between 2 and 120 days.

In an embodiment, stimulating the ICP includes culturing the ICP duringa period of between 3 and 60 days.

In an embodiment, stimulating the ICP includes culturing the ICP in aculture medium including less than 10% serum, for a duration of between1 and 120 days.

In an embodiment, stimulating the ICP includes culturing the ICP in aculture medium including at least 10% serum, for a duration of between 1and 120 days.

In an embodiment, the method includes characterizing the PCP asincluding angiogenic cell precursors (ACPs), in response to anevaluation of at least a feature selected from the group consisting of:a phenotypical feature of cells in the PCP, a genotypical feature ofcells in the PCP, and a physiological feature of cells in the PCP.

In an embodiment, characterizing the PCP includes characterizing the PCPin response to an evaluation of at least two of the features.

In an embodiment, characterizing the PCP includes characterizing the PCPin response to an evaluation of each of the features.

In an embodiment:

the phenotypical feature includes a morphological feature selected fromthe group consisting of: a cell size larger than 20 μm, an elongatedcell, a spindle-shaped cell, an irregularly-shaped cell, a granulatedcell, a cell including an enlarged dark nucleus, a multinuclear cell, acell including flagella-like structures, a cell including pseudopodia,and a cell having a polygonal shape; and

characterizing the PCP includes characterizing the PCP in response to anevaluation of the selected morphological feature.

In an embodiment, characterizing the PCP includes identifying that atleast 1.5% of cells of the PCP have the selected feature.

In an embodiment, characterizing the PCP includes characterizing the PCPin response to an identification in the PCP of a feature selected fromthe group consisting of: CD31Bright, CD34, CD117, CD133, Tie-2,CD34+CD133+, KDR, CD34+KDR+, CD144, von Willebrand Factor, SH2 (CD105),SH3, fibronectin, collagen type I, collagen type III, collagen type IV,ICAM type 1, ICAM type 2, VCAM1, vimentin, BMP-R IA, BMP-RIL CD44,integrin b1, aSM-actin, MUC18, and CXCR4.

In an embodiment, characterizing the PCP includes identifying that atleast 1.5% of cells of the PCP have the selected feature.

In an embodiment, characterizing the PCP includes characterizing the PCPin response to an assessment of uptake by the PCP of Ac-LDL.

In an embodiment, characterizing the PCP includes identifying that atleast 1.5% of cells of the PCP demonstrate uptake of Ac-LDL.

In an embodiment, characterizing the PCP includes identifying that atleast 1.5% of cells that are CD31Bright demonstrate uptake of Ac-LDL.

In an embodiment, characterizing the PCP includes assessing secretion bythe PCP of a molecule selected from the group consisting of: IL-8,angiogenin, VEGF, MMP2, and MMP9.

In an embodiment, characterizing the PCP includes identifying that atleast 1.5% of cells of the PCP secrete the selected molecule.

In an embodiment, characterizing the PCP includes culturing a portion ofthe PCP on a semi-solid extracellular matrix (ECM), and identifying inthe cultured portion a feature selected from the group consisting of: atube-like structure, a colony, a cluster, and a tendency to migratetowards a chemoattractant.

In an embodiment, characterizing the PCP includes identifying that atleast 1.5% of cells in the cultured portion have a property selectedfrom the group consisting of: formation of a tube-like structure, anability to form a colony, a cluster, and a tendency to migrate towards achemoattractant.

In an embodiment, the method includes including identifying the PCP asbeing suitable for therapeutic implantation in response to an assessmentthat the PCP includes at least 1 million ACPs.

In an embodiment, the method includes characterizing the PCP asincluding a cardiomyocyte (CMC) PCP in response to an evaluation of afeature selected from the group consisting of: a phenotypic feature ofcells in the PCP, a genotypic feature on the cells in the PCP, and aphysiological feature of cells in the PCP.

In an embodiment, characterizing the PCP includes characterizing the PCPin response to an evaluation of at least two of the features.

In an embodiment, the method includes characterizing the PCP includescharacterizing the PCP in response to an evaluation of each of thefeatures.

In an embodiment, the phenotypic feature includes a morphologicalfeature selected from the group consisting of: a cell size larger than20 μm, an elongated cell, an irregularly-shaped cell, a granulated cell,a cell including an enlarged dark nucleus, a multinuclear cell, a cellwith dark cytoplasm, and cells arranged in parallel to each other; and

wherein characterizing the PCP includes characterizing the PCP inresponse to an evaluation of the selected morphological feature.

In an embodiment, characterizing the PCP includes characterizing the PCPin response to an identification in the PCP of a feature selected fromthe group consisting of: CD31, CD117, sarcomeric alpha-actin,beta-actin, alpha-actinin, desmin, cardiac troponin T, Connexin-43,alpha/beta-MHC, sarcomeric alpha-tropomyosin, Troponin I, GATA-4,Nkx2.5/Csx, MLC-2, and MEF-2.

In an embodiment, characterizing the PCP includes characterizing the PCPin response to an identification of the PCP as expressing a gene for afactor selected from the group consisting of sarcomeric alpha-actin,beta-actin, alpha-actinin, desmin, cardiac troponin T, Connexin-43,alpha/beta-MHC, sarcomeric, alpha-tropomyosin, Troponin I, GATA-4,Nkx2.5/Csx, MLC-2 and MEF-2.

In an embodiment, the method includes identifying the PCP as beingsuitable for therapeutic implantation in response to an assessment thatthe PCP includes at least 1 million CMC progenitors.

In an embodiment, characterizing the PCP includes identifying that atleast 1.5% of cells of the PCP have a characteristic selected from thegroup consisting of a CMC-progenitor morphological characteristic,expression of a CMC-associated cellular marker, expression of aCMC-progenitor gene product, and expression of a CMC-progenitorphysiological feature.

In an embodiment, characterizing the PCP includes characterizing the PCPin response to an identification in the PCP of an action in response toactivation of the PCP, the action selected from the group consisting of:increasing intracellular Ca2⁺ level, generating membranalelectrophysiological action potentials, and mechanical cellularcontraction in vitro.

In an embodiment, the method includes activating the PCP to produce theselected action, using a technique selected from the group consistingof: electrical activation of the PCP, and chemical activation of thePCP.

In an embodiment, the method includes:

assessing a phenotypic aspect of the PCP and a genotypic aspect of thePCP and a physiological aspect of the PCP; and

designating the PCP as being suitable for implantation in a patient inresponse to each of the assessments.

In an embodiment, assessing the phenotypic aspect of the PCP includesassessing an aspect of the PCP selected from the group consisting of:morphology of the PCP, a cellular marker of cells of the PCP, an enzymeof the PCP, a coenzyme of the PCP, and presence of a designated cellularreceptor on cells of the PCP.

In an embodiment, assessing the genotypic aspect of the PCP includesassessing an aspect of the PCP selected from the group consisting of:production of a gene by cells of the PCP, expression of a gene by cellsof the PCP, and generation of a gene product by cells of the PCP.

In an embodiment, assessing the physiological aspect of the PCP includesassessing an aspect of the PCP selected from the group consisting of:secretion of soluble molecules by cells of the PCP, generation ofsignals by cells of the PCP, response by cells of the PCP to signals,and an enzymatic reaction performed by cells of the PCP.

In an embodiment, the method includes facilitating a diagnosisresponsive to stimulating the ICP to differentiate into the PCP.

In an embodiment, facilitating the diagnosis includes assessing anextent to which the stimulation of the ICP produces a particularcharacteristic of the PCP.

In an embodiment, the method includes transfecting a gene into the ICPprior to stimulating the ICP.

In an embodiment, transfecting the gene includes transfecting into theICP a gene identified as suitable for gene therapy.

In an embodiment, the method includes preparing, as a product foradministration to a patient, the PCP generated by differentiation of theICP into which the gene has been transfected.

In an embodiment, the method includes stimulating the ICP includesincubating the ICP in a container having a surface including agrowth-enhancing factor.

In an embodiment, the method includes the growth-enhancing factor isselected from the group consisting of: collagen, plasma, fibronectin, agrowth factor, tissue-derived extra cellular matrix, and an antibody toa stem cell surface receptor.

In an embodiment, stimulating the ICP includes incubating the ICP in acontainer with a surface including a growth-enhancing molecule otherthan collagen or fibronectin.

In an embodiment, incubating the ICP includes incubating the ICP in acontainer having a surface that includes, in addition to thegrowth-enhancing molecule, at least one of: collagen and fibronectin.

In an embodiment, the method includes mixing the growth-enhancingmolecule with the at least one of: collagen and fibronectin.

In an embodiment, the method includes applying to the surface a layerthat includes the growth-enhancing molecule and a separate layer thatincludes the at least one of: collagen and fibronectin.

In an embodiment, stimulating the ICP includes:

during a low-serum time period, culturing the ICP in a culture mediumincluding less than 10% serum; and

during a high-serum time period, culturing the ICP in a culture mediumincluding greater than or equal to 10% serum.

In an embodiment, culturing the ICP during the low-serum time periodincludes culturing the ICP for a duration of between 1 and 60 days.

In an embodiment, culturing the ICP during the low-serum time periodincludes culturing the ICP for a duration of between 1 and 5 days.

In an embodiment, culturing the ICP during the high-serum time periodincludes culturing the ICP for a duration of between 1 and 120 days.

In an embodiment, culturing the ICP during the high-serum time periodincludes culturing the ICP for a duration of between 1 and 60 days.

In an embodiment, culturing the ICP during the low-serum time period isperformed prior to culturing the ICP during the high-serum time period.

In an embodiment, culturing the ICP during the low-serum time period isperformed following culturing the ICP during the high-serum time period.

In an embodiment, the method includes:

during a hypoxic time period lasting at least 2, hours, culturing theICP under hypoxic conditions; and

during a non-hypoxic time period lasting at least 1 day, culturing theICP under non-hypoxic conditions.

In an embodiment, the hypoxic and non-hypoxic time-periods are within aculturing time period lasting less than 30 days, and wherein culturingthe ICP under hypoxic conditions includes culturing the cells underhypoxic conditions during a first two days of the culturing time period.

In an embodiment, the hypoxic and non-hypoxic time-periods are within aculturing time period lasting less than 30 days, and wherein culturingthe ICP under hypoxic conditions includes culturing the ICP underhypoxic conditions during a last two days of the culturing time period.

In an embodiment, the hypoxic and non-hypoxic time-periods are within aculturing time period lasting less than 30 days, and wherein culturingthe ICP under hypoxic conditions includes culturing the ICP underhypoxic conditions for at least 2 hours between a first two days and alast two days of the culturing time period.

In an embodiment, culturing the ICP under hypoxic conditions isperformed prior to culturing the ICP under non-hypoxic conditions.

In an embodiment, culturing the ICP under hypoxic conditions isperformed following culturing the ICP under non-hypoxic conditions.

In an embodiment, stimulating the ICP includes:

culturing the ICP in a first container during a first portion of aculturing period;

removing at least some cells of the ICP from the first container at theend of the first portion of the period; and

culturing, in a second container during a second portion of the period,the cells removed from the first container.

In an embodiment, the method includes, subsequently to the step ofculturing in the second container:

culturing the ICP in a primary container during a first portion of anadditional culturing period;

removing at least some cells of the ICP from the primary container atthe end of the first portion of the additional period; and

culturing, in a secondary container during a second portion of theadditional period, the cells removed from the primary container.

In an embodiment, stimulating the ICP includes:

culturing the ICP in a first container during a first portion of aculturing period;

removing cells of the ICP from the first container at the end of thefirst portion of the period; and

culturing, in a second container during a second portion of the period,the cells removed from the first container.

In an embodiment, removing at least some cells of the ICP includesselecting for removal cells that adhere to a surface of the firstcontainer.

In an embodiment, removing at least some cells of the ICP includesselecting for removal cells that do not adhere to a surface of the firstcontainer.

In an embodiment, the first container includes on a surface thereof agrowth-enhancing molecule, and wherein culturing the ICP in the firstcontainer includes culturing the ICP in the first container thatincludes the growth-enhancing molecule.

In an embodiment, the growth-enhancing molecule is selected from thegroup consisting of: collagen, plasma, fibronectin, a growth factor,tissue-derived extra cellular matrix and an antibody to a stem cellsurface receptor.

In an embodiment, the second container includes on a surface thereof agrowth-enhancing molecule, and wherein culturing the ICP in the secondcontainer includes culturing the ICP in the second container thatincludes the growth-enhancing molecule.

In an embodiment, the growth-enhancing molecule is selected from thegroup consisting of: collagen, fibronectin, a growth factor, and anantibody to a stem cell surface receptor.

In an embodiment, stimulating includes culturing the ICP with at leastone factor derived from a sample tissue.

In an embodiment, the method includes preparing a conditioned medium forculturing the ICP therein, the conditioned medium including the factor,the factor being derived from the tissue, the tissue being selected fromthe group consisting of: peripheral nerve tissue, central nervous system(CNS) tissue, retinal tissue, pigment epithelial tissue, photoreceptortissue, fetal retinal tissue, embryonic retinal tissue, mature retinaltissue, blood vessel tissue, cardiac tissue, pancreatic endocrinetissue, pancreatic exocrine tissue, smooth muscle tissue, lymphatictissue, hepatic tissue, lung tissue, skin tissue, exocrine glandulartissue, mammary gland tissue, endocrine glandular tissue, thyroid glandtissue, pituitary gland tissue, and plant tissue.

In an embodiment, stimulating includes co-culturing the ICP with asample tissue.

In an embodiment, co-culturing includes preparing the sample tissue by amethod selected from the group consisting of: slicing the sample tissue,and homogenizing the sample issue.

In an embodiment, co-culturing includes:

utilizing the sample tissue to produce a conditioned medium; and

co-culturing the ICP with the sample tissue in the conditioned medium.

In an embodiment, co-culturing includes separating the sample tissuefrom the ICP by a semi-permeable membrane.

In an embodiment, the method includes designating the sample tissue toinclude a tissue selected from the group consisting of: peripheral nervetissue, central nervous system (CNS) tissue, retinal tissue, pigmentepithelial tissue, photoreceptor tissue, fetal retinal tissue, embryonicretinal tissue, mature retinal tissue, blood vessel tissue, cardiactissue, pancreatic endocrine tissue, pancreatic exocrine tissue, smoothmuscle tissue, lymphatic tissue, hepatic tissue, lung tissue, skintissue, exocrine glandular tissue, mammary gland tissue, endocrineglandular tissue, thyroid gland tissue, pituitary gland tissue, andplant tissue.

There is further provided, in accordance with an embodiment of theinvention, a method for treating a patient, including:

identifying a patient having a sexual dysfunction; and

administering angiogenic cell precursors to the patient, in order totreat the dysfunction.

There is also provided, in accordance with an embodiment of the presentinvention, a method including in vitro stimulating a core cellpopulation (CCP) of at least 5 million cells that have a density of lessthan 1.072 g/ml, and at least 1% or at least 2% of which areCD34+CD45−/Dim, to differentiate into a progenitor/precursor cellpopulation (PCP).

For some applications, the CCP includes at least 5 million cells thathave a density of less than 1.062 g/ml, at least 2% of which areCD34+CD45−/Dim, and stimulating the CCP includes stimulating the CCPthat has the at least 5 million cells that have a density of less than1.062 g/ml.

For some applications, the method includes preparing the PCP as aproduct for administration to a patient. Alternatively, the methodincludes preparing the PCP as a research tool or a diagnostic tool.

For some applications, stimulating the CCP includes only stimulating theCCP if the CCP is derived from a mammalian donor. For some applications,the method includes applying cells extracted from a mammalian donor toone or more gradients suitable for selecting cells having a density lessthan 1.072 g/ml, and deriving the CCP from the cells applied to thegradient.

For some applications, the CCP is characterized by at least 2.5% of theCCP being CD34+CD45−/Dim, and stimulating the CCP includes stimulatingthe CCP having the at least 2.5% of the CCP that are CD34+CD45−/Dim. Forsome applications, the CCP is characterized by at least 50% of the CCPbeing CD31Bright, and stimulating the CCP includes stimulating the CCPhaving the at least 50% of the CCP that are CDC31Bright+. For someapplications, the CCP is characterized by at least 40% of the CCP beingCD31Bright, and stimulating the CCP includes stimulating the CCP havingthe at least 40% of the CCP that are CD31Bright.

For some applications, stimulating the CCP includes stimulating the CCPto differentiate into a pre-designated, desired class of progenitorcells.

For some applications, stimulating the CCP includes culturing the CCPduring a period of between 3 and 30, 60, or 120 days.

For some applications, the method includes deriving the CCP from atleast one source selected from the group consisting of: embryonictissue, fetal tissue, umbilical cord blood, umbilical cord tissue,neonatal tissue, adult tissue, bone marrow, mobilized blood, peripheralblood, peripheral blood mononuclear cells, skin cells, and plant tissue.Alternatively, the method includes deriving the CCP from at least onesource selected from the group consisting of: fresh tissue and frozentissue. For some applications, the method includes identifying anintended recipient of the PCP, and deriving the CCP from at least onesource selected from the group consisting of: tissue autologous totissue of the intended recipient, tissue syngeneic to tissue of theintended recipient, tissue allogeneic to tissue of the intendedrecipient, and tissue xenogeneic to tissue of the intended recipient.

For some applications, stimulating the CCP includes incubating the CCPin a container having a surface including an antibody.

For some applications, stimulating the CCP includes incubating the CCPin a container having a surface including a plasma.

For some applications, stimulating the CCP includes culturing the CCPfor a period lasting between 1 and 5, 10, or 20 days in a culture mediumincluding less than 5% serum. For some applications, stimulating the CCPincludes culturing the CCP for a period lasting between 1 and 5, 10, or20 days in a culture medium including at least 10% serum.

For some applications, stimulating the CCP includes culturing the CCP inthe presence of at least one of the following: erythropoietin, a statin,an antidiabetic agent, a thiazolidinedione, rosiglitazone, aproliferation-differentiation-enhancing agent, anti-CD34, anti-Tie-2,anti-CD133, anti-CD117, LIF, EPO, IGF, b-FGF, M-CSF, GM-CSF, TGF alpha,TGF beta, VEGF, BHA, BDNF, NGF, NT3, NT4/5, GDNF, S-100, CNTF, EGF,NGF3, CFN, ADMIF, prolactin, an adrenocorticoid hormone, ACTH,glutamate, serotonin, acetylcholine, NO, retinoic acid (RA) or any othervitamin D derivative, heparin, insulin, forskolin, cortisone, cortisol,dexamethasone, estrogen, a steroid, MCDB-201, MCT-165, glatirameracetate, a glatiramer acetate-like molecule, IFN alpha, IFN beta or anyother immunoregulatory agent, sodium selenite, linoleic acid, ascorbicacid, transferrin, 5-azacytidine, PDGF, VEGF, cardiotrophin, andthrombin.

For some applications, the method includes preparing the CCP, andfacilitating a diagnosis responsive to a characteristic of thepreparation of the CCP.

For some applications, the method includes freezing the CCP prior tostimulating the CCP. For some applications, the method includes freezingthe PCP.

For some applications, the method includes transporting the CCP to asite at least 10 km from a site where the CCP is first created, andstimulating the CCP at the remote site. For some applications, themethod includes transporting the PCP to a site at least 10 km from asite where the PCP is first created.

In an embodiment, the method includes facilitating a diagnosisresponsive to stimulating the CCP to differentiate into the PCP. Forsome applications, facilitating the diagnosis includes assessing anextent to which the stimulation of the CCP produces a particularcharacteristic of the PCP.

In an embodiment, the method includes transfecting a gene into the CCPprior to stimulating the CCP. For some applications, the method includespreparing, as a product for administration to a patient, the PCPgenerated by differentiation of the CCP into which the gene has beentransfected.

In an embodiment, the method includes transfecting a gene into the PCPprior to administration of the PCP to a patient.

In an embodiment, stimulating the CCP includes incubating the CCP in acontainer with a surface including a growth-enhancing molecule otherthan collagen or fibronectin. For some applications, incubating the CCPcells includes incubating the CCP in a container having a surface thatincludes, in addition to the growth-enhancing molecule, at least one ofcollagen, plasma and fibronectin. For some applications, the methodincludes mixing the growth-enhancing molecule with the at least one of:collagen, plasma and fibronectin. For some applications, the methodincludes applying to the surface a layer that includes thegrowth-enhancing molecule and a separate layer that includes the atleast one of: collagen, plasma and fibronectin.

In an embodiment, stimulating the. CCP includes:

during a low-serum time period, culturing the CCP in a culture mediumincluding less than 10% serum; and

during a high-serum time period, culturing the CCP in a culture mediumincluding greater than or equal to 10% serum.

For some applications, culturing the CCP during the low-serum timeperiod includes culturing the CCP for a duration of between 1 and 5 or20 days. For some applications, culturing the CCP during the high-serumtime period includes culturing the CCP for a duration of between 1 and30, 60, or 120 days. For some applications, culturing the CCP during thelow-serum time period is performed prior to culturing the CCP during thehigh-serum time period. For some applications, culturing the CCP duringthe low-serum time period is performed following culturing the CCPduring the high-serum time period.

In an embodiment, the method includes:

during a hypoxic time period lasting at least 2 hours, culturing the CCPunder hypoxic conditions; and

during a non-hypoxic time period lasting at least 1 day, culturing theCCP under non-hypoxic conditions.

For some applications, the hypoxic and non-hypoxic time-periods arewithin a culturing time period lasting less than 120 days (e.g., lessthan 30 days), and culturing the CCP under hypoxic conditions includesculturing the cells under hypoxic conditions during a first two days ofthe culturing time period. For some applications, the hypoxic andnon-hypoxic time-periods are within a culturing time period lasting lessthan 120 days (e.g., less than 30 days), and culturing the CCP underhypoxic conditions includes culturing the CCP under hypoxic conditionsduring a last two days of the culturing time period. For someapplications, the hypoxic and non-hypoxic time-periods are within aculturing time period lasting less than 120 days (e.g., less than 30days), and culturing the CCP under hypoxic conditions includes culturingthe CCP under hypoxic conditions for at least 2 hours between a firsttwo days and a last two days of the culturing time period.

For some applications, culturing the CCP under hypoxic conditions isperformed prior to culturing the CCP under non-hypoxic conditions.Alternatively, culturing the CCP under hypoxic conditions is performedfollowing culturing the CCP under non-hypoxic conditions.

In an embodiment, stimulating the CCP includes:

culturing the CCP in a first container during a first portion of aculturing period;

removing all or at least some cells of the CCP from the first containerat the end of the first portion of the period; and

culturing, in a second container during a second portion of the period,the cells removed from the first container.

For some applications, removing at least some cells of the CCP includesselecting for removal cells that adhere to a surface of the firstcontainer. For some applications, removing at least some cells of theCCP includes selecting for removal cells that do not adhere to a surfaceof the first container.

For some applications, the first container includes on a surface thereofa growth-enhancing molecule, and culturing the CCP in the firstcontainer includes culturing the CCP in the first container thatincludes the growth-enhancing molecule.

For some applications, the growth-enhancing molecule is selected fromthe group consisting of: collagen, plasma, fibronectin, a growth factor,tissue-derived extra cellular matrix and an antibody to a stem cellsurface receptor.

For some applications, the second container includes on a surfacethereof a growth-enhancing molecule, and culturing the CCP in the secondcontainer includes culturing the CCP in the second container thatincludes the growth-enhancing molecule.

For some applications, the growth-enhancing molecule is selected fromthe group consisting of collagen, plasma, fibronectin, a growth factor,tissue-derived extra cellular matrix and an antibody to a stem cellsurface receptor.

In an embodiment, stimulating includes culturing the CCP with at leastone factor derived from a target tissue. For some applications, themethod includes preparing a conditioned medium for culturing the CCPtherein, the conditioned medium including the factor, the factor beingderived from a tissue selected from the group consisting of: peripheralnerve tissue, central nervous system (CNS) tissue, retinal tissue,pigment epithelial tissue, photoreceptor tissue, fetal retinal tissue,embryonic retinal tissue, mature retinal tissue, blood vessel tissue,cardiac tissue, pancreatic endocrine tissue, pancreatic exocrine tissue,smooth muscle tissue, lymphatic tissue, hepatic tissue, lung tissue,skin tissue, exocrine glandular tissue, mammary gland tissue, endocrineglandular tissue, thyroid gland tissue, pituitary gland tissue, andplant tissue.

In an embodiment, stimulating includes co-culturing the CCP with atissue. For some applications, co-culturing includes preparing a targettissue by a method selected from the group consisting of: slicing thetarget tissue, and homogenizing the target issue. For some applications,co-culturing includes utilizing the target tissue to produce aconditioned medium, and co-culturing the CCP with the target tissue inthe conditioned medium. For some applications, co-culturing includesseparating the target tissue from the CCP by a semi-permeable membrane.

For some applications, the method includes designating a tissue forco-culture purposes to include a tissue selected from the groupconsisting of: peripheral nerve tissue, central nervous system (CNS)tissue, retinal tissue, pigment epithelial tissue, photoreceptor tissue,fetal retinal tissue, embryonic retinal tissue, mature retinal tissue,blood vessel tissue, cardiac tissue, pancreatic endocrine tissue,pancreatic exocrine tissue, smooth muscle tissue, lymphatic tissue,hepatic tissue, lung tissue, skin tissue, exocrine glandular tissue,mammary gland tissue, endocrine glandular tissue, thyroid gland tissue,pituitary gland tissue, and plant tissue.

There is also provided, in accordance with an embodiment of the presentinvention, a method including in vitro stimulating an elemental cellpopulation (ECP) of at least 5 million cells that have a density of lessthan 1.072 g/ml, at least 1.5% of which are CD34+CD45−/Dim, and at least30% of which are CD31Bright, to differentiate into aprogenitor/precursor cell population (PCP).

For some applications, the present invention includes treating a patientwith a PCP administrated systemically.

For some applications, the present invention includes treating a patientwith a PCP administrated locally to injured tissue.

There is also provided, in accordance with an embodiment of the presentinvention, a method for treating a patient including administering a PCPusing an implantable medical device, which, in an embodiment, includesmetal, plastic, glass, or another material, and which for someapplications is biodegradable. As appropriate for a given application,the medical device may include a stent, microparticles, ormicrocapsules.

There is also provided, in accordance with an embodiment of the presentinvention, a method comprising implanting, at a site including injuredtissue, a medical device including a PCP, to enable increased survivalat the site of the PCP.

There is also provided, in accordance with an embodiment of the presentinvention, a method including a medical device that provides slowrelease of a PCP into injured tissue.

There is also provided, in accordance with an embodiment of the presentinvention, a method including coupling a PCP to a medical device,wherein the PCP is adapted to be a source of therapeutic solublemolecules to a subject in whom the medical device is implanted. For someapplications, apparatus comprises a chamber having disposed therein apopulation of stem cells (e.g., a PCP produced using techniquesdescribed herein), the chamber being surrounded by a semi-permeablemembrane. Therapeutic molecules leave the chamber through the membrane,and treat the patient. As appropriate, techniques and apparatusdescribed in the above-referenced US Patent Application Publication2005/0209556 to Tresco and article by Rehman may be practiced incombination with this embodiment, mutatis mutandis.

There is also provided, in accordance with an embodiment of the presentinvention, a method including attaching a PCP to a medical device,wherein the medical device is a source of soluble molecules that supportthe PCP.

There is additionally provided, in accordance with an embodiment of thepresent invention, a composition of matter, including a population ofcultured cells that includes a sub-population of cells that both stainas CD31Bright and demonstrate uptake of Ac-LDL+.

In an embodiment, the sub-population secretes IL-8.

In an embodiment, the sub-population secretes at least 50 pg IL-8 per10⁶ cells/ml over a period of at least 24 hours.

In an embodiment, the sub-population secretes at least 150 pg IL-8 per10⁶ cells/nal over a period of at least 24 hours.

In an embodiment, the sub-population secretes at least 1000 pg per 10⁶cells/ml over a period of at least 24 hours.

In an embodiment, at least 1.5% of the cells of the population secrete amolecule selected from the group consisting of: IL-8, angiogenin, VEGF,MMP2, and MMP9.

In an embodiment, at least 1.5% of the cells of the population have atendency to migrate toward a chemoattractant selected from the groupconsisting of: bFGF, VEGF, SCF, G-CSF, GM-CSF, SDF-1, and IL-8.

There is further provided, in accordance with an embodiment of thepresent invention, a composition of matter, including a population ofcultured cells that includes a sub-population of cells that stain asCD31Bright, demonstrate uptake of Ac-LDL+ and secrete interleukin-8.

In an embodiment, the sub-population includes at least 10% of the cellsin the population.

In an embodiment, the sub-population includes at least 25% of the cellsin the population.

In an embodiment, the sub-population includes at least 50% of the cellsin the population.

In an embodiment, the sub-population secretes at least 50 pg IL-8 per10⁶ cells/ml over a period of at least 24 hours.

In an embodiment, the sub-population secretes at least 150 pg IL-8 per10⁶ cells/ml over a period of at least 24 hours.

In an embodiment, the sub-population secretes at least 1000 pg IL-8 per10⁶ cells/ml over a period of at least 24 hours.

In an embodiment, at least 1.5% of the cells of the population include amorphological feature selected from the group consisting of: a cell sizelarger than 20 um, an elongated cell, a spindle-shaped cell, anirregularly-shaped cell, a granulated cell, a cell including an enlargeddark nucleus, a multinuclear cell, a cell including flagella-likestructures, a cell including pseudopodia, and a cell having a polygonalshape.

In an embodiment, at least 1.5% of the cells of the population include afeature selected from the group consisting of CD34, CD117, CD133, Tie-2,CD34+CD133+, KDR, CD34+KDR+, CD144, von Willebrand Factor, SH2 (CD105),SH3, fibronectin, collagen type I, collagen type III, collagen type IV,ICAM type 1, ICAM type 2, VCAM1, vimentin, BMP-R IA, BMP-RII, CD44,integrin b1, aSM-actin, MUC18, CXCR4 and CXCR8.

In an embodiment, at least 1.5% of the cells of the population secrete amolecule selected from the group consisting of angiogenin, VEGF, MMP2,and MMP9.

In an embodiment, at least 1.5% of the cells of the population include afeature selected from the group consisting of: a tube-like structure, atendency to form a colony, a tendency to form a cluster, and a tendencyto migrate towards chemoattractants selected from the group consistingof: bFGF, VEGF, SCF, G-CSF, GM-CSF, SDF-1, and IL-8.

In an embodiment, characterizing the PCP includes culturing at least aportion of the PCP on a surface, and identifying a tendency of the atleast a portion of the PCP to migrate toward IL-8.

The present invention will be more fully understood from the followingdetailed description of embodiments thereof, taken together with thedrawings, in which:

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing results obtained from CCP cells in onerepresentative experiment, in accordance with an embodiment of thepresent invention;

FIG. 2 is a photograph showing morphology of angiogenic cell precursorcells, produced in accordance with an embodiment of the presentinvention;

FIG. 3 is a photograph characterizing uptake of Ulex-Lectin and stainingwith CD31 stain of an ACP-rich PCP, produced in accordance with anembodiment of the present invention;

FIG. 4 is a photograph characterizing uptake of Ac-LDL and staining withCD31 stain of an ACP-rich PCP, produced in accordance with an embodimentof the present invention;

FIG. 5 is a photograph showing tube formation in an ACP-rich PCP,produced in accordance with an embodiment of the present invention;

FIGS. 6A and 6B are graphs showing migration of Ac-LDL-DiO pre-labeledACPs in response to hIL-8, in accordance with an embodiment of thepresent invention;

FIG. 7 is a graph showing migration of Ac-LDL-DiO pre-labeled ACPs inresponse to a cultured medium, in accordance with an embodiment of thepresent invention;

FIG. 8 is a graph of migration of PBMC cells in response to hIL-8, inaccordance with an embodiment of the present invention;

FIGS. 9A and 9B are graphs showing experimental results of improvedejection fraction and reduced necrosis in response to injection of ACPcells in accordance with an embodiment of the present invention;

FIGS. 9C, 9D, and 9E are photographs showing sections taken from a rat'sheart after injection of ACPs derived from a human-PBMC-derived CCP,produced in accordance with an embodiment of the present invention;

FIG. 10 is a photograph showing the morphology of cardiomyocytes derivedfrom the CCP and produced in accordance with an embodiment of thepresent invention;

FIGS. 11A, 11B, and 11C are photographs showing immunostaining ofCCP-derived cardiomyocytes, in accordance with an embodiment of thepresent invention;

FIGS. 12A and 12B are graphs showing flow cytometry analysis results,obtained from immunostaining of a cardiomyocyte-rich PCP, in accordancewith an embodiment of the present invention; and

FIG. 13 is a graph showing experimental results of improved ejectionfraction in a rat model of acute myocardial infarction followinginjection of the CCP-derived cardiomyocytes, in accordance with anembodiment of the present invention.

DETAILED DESCRIPTION OF EMBODIMENTS Example 1

A test was carried out in accordance with an embodiment of the presentinvention, and results are shown in Table 1 below. Peripheral blood wasextracted from ten human volunteers for use in ten respectiveexperiments. In each experiment, cells were fractioned from the bloodusing a Ficoll™ gradient in order to generate a population of peripheralblood mononuclear (PBMC) cells as source cells (“S. cells”).Subsequently, a CCP was generated in accordance with protocols describedherein for Percoll (TM) based enrichment. Results in Table 1 showenrichment of the percentages of CD34+CD45−/Dim cells in the CCPcompared to the source cells. Enrichment is defined as the percentage ofcells having a given characteristic in the CCP, divided by thepercentage of cells having that characteristic in the source cells.

TABLE 1 % CD34+ % Viability % CD45 CD45−/Dim Exp S. S. Enrichment Nocells CCP cells CCP S. cells CCP factor 1 97.56 97.86 94.00 93.46 1.44.07 2.9 2 98.49 97.61 92.09 87.10 0.77 3.48 4.5 3 94.28 100 94.72 96.440.72 2.31 3.2 4 98.82 98.18 93.11 92.77 0.24 2.69 11.2 5 98.10 98.5363.15 84.30 1.78 2.77 1.6 6 98.54 98.33 91.58 76.16 0.69 2.37 3.4 798.18 97.78 95.58 94.46 0.88 3.7 4.2 8 99.49 97.93 96.11 92.39 0.83 6.147.4 9 99.09 97.64 96.75 96.55 0.39 2.24 5.7 10  97.53 99.37 84.46 98.440.52 1.67 3.2 AVG 98.01 98.32 90.58 91.41 0.82 3.14 4.7

Example 2

In a separate set of experiments, in accordance with an embodiment ofthe present invention, results were obtained as shown in FIG. 1 andTable 2 below. Peripheral blood was extracted from ten human volunteersfor use in ten experiments. A CCP was generated in accordance withprotocols described herein (see Example 1). Results in FIG. 1 and inTable 2 show the fluorescent intensity of CD31Bright cells in the CCP.CD31 brightness (Dim or Bright) is defined as the ratio betweenintensity resulting from staining using anti-CD31 FITC-conjugatedmonoclonal antibodies and intensity resulting from staining usingisotype control FITC-conjugated antibodies.

FIG. 1 is a graph showing results obtained from CCP cells in onerepresentative experiment, in accordance with an embodiment of thepresent invention. CCP cells stained using isotype controlFITC-conjugated antibodies are represented by the dashed line and CCPcells stained using FITC-conjugated anti-CD31 antibodies are representedby the black line. Three different intensity areas were marked:

(a) M1—low intensity corresponding to non-specific staining of isotypecontrol or cells that do not express CD31, located at geometric meanintensity of 5.38;

(b) M2—dim intensity corresponding to cells expressing CD31 at ageometric mean intensity of 46.69; and

(c) M3—bright intensity corresponding to cells expressing CD31 at ageometric mean intensity of 478.45.

Table 2 is a numerical summary of intensities M1, M2 and M3 and theirrespective ratios resulting from ten independent experiments.

TABLE 2 CD31 Intensity Geo Mean Isotype EXP Control dim bright IntensityRatio No. M1 M2 M3 M2/M1 M3/M1 M3/M2 1 5.25 42.90 299.92 8 57 7 2 5.3846.69 478.45 9 89 10 3 5.52 30.37 340.24 6 62 11 4 4.9 28.41 266.46 6 549 5 4.57 33.19 456.80 7 100 14 6 5.31 34.94 384.76 7 72 11 7 2.91 25.45318.20 9 109 13 8 2.19 27.43 361.86 13 165 13 9 3.86 33.57 310.46 9 80 910  5.3 42.68 400.03 8 75 9 AVG 4.52 34.56 361.72 8.00 86.50 10.69 SE0.37 2.30 21.74 0.63 10.42 0.66CD31Bright cells' (M3) mean intensity is 86.5 (SE=10.42) times greaterthan the negative control intensity (M1) and 10.69 (SE=0.66) times morethan CD31Dim cells (M2) (which themselves have an intensity 8.00(SE=0.63) times more than M1). Thus, results indicate that the CCP wasenriched to provide CD31+ cells.

Example 3

In a separate set of experiments, in accordance with an embodiment ofthe present invention, results were obtained as shown in Table 3 below.Peripheral blood was extracted from nine human volunteers for use innine experiments. A CCP was generated in accordance with protocolsdescribed hereinabove with reference to Example 1.

TABLE 3 % CD31Bright Enrichment Exp No. S. Cells CCP Factor 1 10.1 60.46.0 2 25.4 80.85 3.2 3 19.1 76.85 4.0 4 25.1 77.3 3.1 5 16.1 75.8 4.7 612.7 75.0 5.9 7 17.5 53.3 3.1 8 21.9 80.96 3.7 9 18.6 64.58 3.5 AVG 18.571.67 4.13Results in Table 3 indicate percentage enrichment of CD31Bright cells inthe CCP as compared to the source cells.

Example 4

In a separate set of experiments, a human-PBMC-derived CCP was culturedin order to generate an ACP-rich PCP; the CCP was grown on fibronectinor plasma-coated T75 flasks in the presence of medium containingautologous serum (>=10%), 2 ng/ml VEGF, and 5 IU/ml Heparin.

FIG. 2 is a photograph showing the morphology of a typical angiogeniccell precursor (ACP) population, produced in the experiments of Example3, in accordance with an embodiment of the present invention. Typically,elongated and spindle-shaped cells are observed in cultures of ACPs.This image was obtained from ×200 magnification of cultured ACPs.

Example 5

In a separate set of experiments, a human-PBMC-derived CCP was culturedin order to generate an ACP-rich PCP, as described hereinabove withrespect to Example 4. The CCP was grown on fibronectin or plasma-coatedT75 flasks in the presence of medium containing autologous serum(>=10%), 2 ng/ml VEGF, and 5 IU/ml Heparin.

FIG. 3 contains photographs of a typical angiogenic cell precursor (ACP)population, produced in the experiments of Example 2, in accordance withan embodiment of the present invention. Harvested cells were loaded on aglass slide and fixed prior to their specific staining. Stained cellswere mounted using a fluorescent mounting solution containing thenuclear stain DAPI. Figures A1-A3 are a series of photographs from cellsstained with FITC-conjugated Ulex-Lectin, cells stained withPE-conjugated anti-CD31, or cells that stained with both Ulex-Lectin andanti-CD31, in accordance with respective embodiments of the presentinvention. A1 is a photograph of cells stained with the nuclear markerDAPI. A2 is a photograph showing green emission resulting from stainingof the same cells with FITC-conjugated Ulex-lectin. A3 is a photographshowing red emission resulting from staining of the same cells withPE-conjugated anti-CD31 antibodies. Figures B1-B3 are a series ofphotographs from cells stained with isotype control antibodies, inaccordance with respective embodiments of the present invention. B1 is aphotograph of cells stained with the nuclear marker DAPI, B2 is aphotograph showing green emission resulting from staining of the samecells with FITC-conjugated mouse IgG antibodies, and B3 is a photographshowing red emission resulting from staining of the same cells withPE-conjugated mouse IgG Antibodies.

Typically, ACP cells fluoresce both red and green indicating adhesion ofboth Ulex-Lectin and anti-CD31 thereto. Images were obtained from ×200magnification.

Example 6

In a separate set of experiments, a human-PBMC-derived CCP was culturedin order to generate an ACP-rich PCP, as described hereinabove withreference to Example 4. The CCP was grown on fibronectin orplasma-coated T75 flasks in the presence of medium containing autologousserum (>=10%), 2 ng/ml VEGF, and 5 IU/ml Heparin.

FIG. 4 contains photographs of a typical angiogenic cell precursor (ACP)population, produced in the experiments of Example 4, in accordance withan embodiment of the present invention. Harvested cells were loaded on aglass slide and fixed prior to their specific staining. Stained cellswere mounted with a fluorescent mounting solution containing the nuclearstain DAPI. Figures A2-A3 are a series of photographs demonstratinguptake of Ac-LDL, cells stained with anti-CD31, or cells that show bothuptake of Ac-LDL and staining with anti-CD31, in accordance withrespective embodiments of the present invention. A1 is a photograph ofcells stained with the nuclear marker DAN, A2 is a photograph showinggreen emission resulting from uptake of FITC labeled-Ac-LDL by the samecells, and A3 is a photograph showing red emission resulting fromstaining of the same cells with PE-conjugated anti-CD31 antibodies.Figures B1-B3 are a series of photographs from cells stained withisotype control antibodies, in accordance with an embodiment of thepresent invention. B1 is a photograph of cells stained with the nuclearmarker DAPI, B2 is a photograph showing green emission resulting fromstaining of the same cells with FITC-conjugated mouse IgG antibodies,and A3 is a photograph showing red emission resulting from staining ofthe same cells with PE-conjugated mouse IgG Antibodies.

Typically, ACP cells fluoresce both green and red indicating that ACPsuptake Ac-LDL as well as comprise CD31. Images were obtained from ×200magnification.

Example 7

In the same set of experiments, the human-PBMC-derived CCP was culturedin order to generate an ACP-rich PCP as described hereinabove withrespect to Example 4. Flow-cytometry percentage staining results fromnine independent experiments are summarized in Table 4, and show theaverage staining results obtained on day 5 of culturing.

TABLE 4 Number experiments Average on Standard (n) day 5 Error % CD34 953.1 6.9 % KDR 9 2.3 1.1 % Tie-2 9 6.6 1.6 % Ac-LDL x CD31Bright 9 60.74.7Results using such a protocol typically yield a PCP having an average of60.7% of cells that both demonstrate uptake of Ac-LDL and stain forCD31Bright.

Example 8

In a separate set of experiments, a human-PBMC-derived CCP was culturedin order to generate an ACP-rich PCP, as described hereinabove withrespect to Example 4. Harvested ACP-rich PCP cells were washed fromculture medium and incubated for 24 hours in a serum-free medium.Average secretion levels (pg/ml) of IL-8, VEGF, and angiogenin asobtained from four independent experiments are summarized in Table 5.

TABLE 5 Group IL-8 pg/ml VEGF pg/ml Angiogenin pg/ml Control Medium ≦20≦20 ≦20 ACP derived 10107 165 615 medium

Example 9

In the same set of experiments, a human-PBMC-derived CCP was cultured inorder to generate an ACP-rich PCP, as described hereinabove withreference to Example 4. Angiogenic pattern and vascular tube formationof ACP-rich PCP cells were examined microscopically following plating ofthe cells on an extracellular matrix gel (ECM). Typically, semi-closedand closed polygons of capillaries and complex mesh-like capillarystructures were observed and scored according to a scale published byKayisli et al. (52) as grade 4-5, indicating the angiogenic-inducingproperties of the ACP-rich PCP.

FIG. 5 is a photograph showing tube formation in an ACPs, produced inthe experiments of Example 6, in accordance with an embodiment of thepresent invention. Typical mesh-like capillary structures generated froma harvested ACPs are present in the culture and are suitable foradministration to a human.

Example 10

In a separate set of experiments, a human-PBMC-derived CCP was culturedin order to generate an ACP-rich PCP; the CCP was grown on fibronectinor plasma-coated T75 flasks in the presence of medium containingautologous serum (>=10%), 2-10 ng/ml VEGF, and 5 IU/ml Heparin. At theend of the culturing period, ACP cells were harvested and labeled with0.8 ug/ml Ac-LDL-DiO for 15 min at 37 C and placed in inserts which wereplaced in wells. One million labeled ACPs were placed on microporousmembrane inserts with a pore size of 8 micrometer. 200 ul medium wasplaced at the bottom of each of the wells. Negative control (M199),positive control (e.g., 20 ng/ml VEGF, 20 ng/ml bFGF, and 20 ng/ml SCF)and 0.08-60 ng/ml human recombinant Interleukin-8 (hIL-8) diluted inM199 medium were plated in respective wells and the ACP cells wereallowed to migrate toward each respective medium. Following 1 hourincubation in the presence of the negative control medium, the positivecontrol medium, and the IL-8 containing media, labeled migrating cellsfrom 10-15 random microscopic fields were evaluated using fluorescentmicroscope and automated counting software (NTH ImageJ). Calculation ofcell number per 1 mm̂2 was based on area of counting field (×20) whichequals 0.178 mm̂2, and each mm̂2 contains 5.62 fields. Assessment of ACPmigratory potential indicated that ACPs migrate toward chemokines suchas VEGF, bFGF, SCF, and hIL-8 in a manner dependent on respectiveconcentrations thereof, e.g., hIL-8 concentration of typically higherthan 6.7 ng/ml induces substantial migration of ACPs, and hIL-8concentrations of about 7-20 ng/ml typically induce substantialmigration of ACPs.

FIGS. 6A and 6B are graphs showing results obtained in five experimentsof Example 10, in accordance with an embodiment of the presentinvention. FIG. 6A shows migration toward negative and positive controlmedia of Ac-LDL-DiO pre-labeled ACPs. FIG. 6B shows a dosage-dependencecurve reflecting migration of Ac-LDL-DiO pre-labeled ACPs in response toincreasing concentrations of hIL-8. ACPs derived from thehuman-PBMC-derived CCP show statistically significant migration towardthe positive control samples. Moreover, ACP migration corresponding toincreasing hIL-8 doses was observed. Dose-dependent ACP migration peakedat 6.7-20 ng/ml of hIL-8.

Example 11

In a separate set of experiments, the human-PBMC-derived CCP wascultured in order to generate an ACP-rich PCP, as described hereinabovewith reference to Examples 4 or 10. In some embodiments, generation ofthe ACP-rich PCP is attributed to migration of ACP cells to a specificchemokine, in combination with the differentiation of CCP cells.Migratory potential of ACP-rich PCP was measured as describedhereinabove with respect to Example 10. In this example, a conditionedmedium (CM) was generated using the patients' cells which secretechemokines into the medium. The patients' cells were then extracted fromthe medium, leaving a chemokine-rich medium for subsequent plating ofACP therein. The potential for

ACP migration in response to chemokines was then assessed when the ACPswere incubated for 1 hour with conditioned medium.

Following 1 hour incubation in the presence of negative control (M199);20 ng/ml hIL-8; or CM (at concentrations of 2-20 ng/ml), migration oflabeled cells from 10-15 random microscopic fields was evaluated using afluorescent microscope and automated counting software (NIH ImageJ).Calculation of cell number per 1 mm̂2 is based on area of counting field(×20)=0.148 mm̂2 and thus each square millimeter contains 6.7 fields. Itwas determined that ACPs migrate toward chemokines secreted during theproduction of the ACP-rich PCP.

For some applications, the generated ACP-rich PCP batches were used totreat cardiovascular patients. All patients treated with these batchesshowed more than 10% improvement in left-ventricular-ejection fractionboth 3 and 6 months following treatment. It is hypothesized that thisimprovement was enabled at least in part by the migration of ACPs to thevicinity.

FIG. 7 is a graph showing results obtained in four experiments ofExample 11, in accordance with an embodiment of the present invention.The ACPs derived from the human-PBMC-derived CCP show statisticallysignificant migration toward the conditioned medium containing secretedchemokines; this medium was generated in the process of the productionof ACP-rich PCP.

Example 12

In a separate set of experiments, migratory potential of human-PBMCtoward hIL-8 was measured. In vitro assessment of PBMC migratorycapability in response to hIL-8 was used to determine the potential ofIL-8 to mobilize blood derived stem/progenitor cells from peripheralblood to locations in which high concentrations of IL-8 are expressed invivo. Peripheral blood was extracted from six human volunteers for usein six respective experiments. In each experiment, a Ficoll™ gradientwas used to generate a population of PBMCs. One million PBMCs wereplaced on 3 um pore size microporous membrane inserts which were placedin wells. 200 ul medium was placed at the bottom of each of the wells.Negative control (M199) and positive control (20 ng/ml hIL-8) diluted inM199 medium were plated in respective wells and the PBMCs cells wereallowed to migrate toward each respective medium. Following 1 hourincubation in the presence of the negative control medium and thepositive control medium, migration of cells from 10-15 randommicroscopic fields was evaluated using a fluorescent microscope andautomated counting software (NIH ImageJ). Calculation of cell number per1 mm̂2 is based on area of counting field (×20) which equals 0.148 mm̂2,and each square millimeter contains 6.7 fields. It was determined thathIL-8 induced mobilization of only a small fraction of the PBMCs,probably the stem/progenitor cells.

FIG. 8 is a graph of migration of PBMCs in response to hIL-8, inaccordance with an embodiment of the present invention. The results wereobtained from six experiments (Example 12), and show thatstem/progenitor cells derived from human-PBMCs migrate toward hIL-8.

Example 13

Reference is now made to FIGS. 9A and 9B which are graphs showingresults obtained in the experiments following injection into rats ofACP-rich PCPs derived from a human-PBMC-derived CCP (as describedhereinabove with respect to Example 4) following acute myocardialinfarction, in accordance with an embodiment of the present invention.

The human-PBMC-derived CCP was cultured in order to generate an ACP-richPCP as described in Example 4. ACP-rich PCP therapeutic potential wasthen assessed in a rat model of acute myocardial infarction which wasinduced in 15 male nude rats (200-225 g) by ligation of the leftanterior descending (LAD) artery. Six days after myocardial infarction,10 rats were injected with 1.5×10̂6 ACP-enriched cells (ACP, n=10), while5 rats were injected with the culture medium (Control, n=5), via theaortic arch. Cardiac function (ejection fraction) and the ratio ofnecrotic scar area to left ventricular free wall area were measured 28days following the ACP-rich PCP and the culture medium administrations.It is to be noted that the percentage of ejection fraction of theACP-administered rats, as represented by FIG. 9A, increasedsubstantially in comparison to the decreased percentage ejectionfraction of the control rats. Additionally, a percentage reduction ofnecrotic tissue was observed in the ACP-administered rats in comparisonto the percentage of necrotic tissue observed in the control rats.Paraffin fixed tissue sections obtained from the 10 ACP-administeredrats were stained in order to trace engrafted human cells andcardiomyocyte (CMC) markers in the border area of the scar tissue.

FIGS. 9C, 9D and 9E are photographs showing typical sections taken froma heart of one of the 10 rats 28 days after the injection of the ACPsderived from a human-PBMC-derived CCP in the experiments of the presentexample (Example 13) in accordance with an embodiment of the presentinvention. FIG. 9C shows staining of the rat's heart cells withanti-human mitochondria. FIG. 9D shows the cells stained for CMC markers(myosin heavy chain (MHC)), FIG. 9E shows the rat heart cells stainedfor cardiac Troponin I. (Reference is again made to FIG. 9C-E. Thestained cells are marked by arrows). These results depicted in FIGS.9C-D demonstrate that the human ACPs, derived in accordance with anembodiment of the present invention from the hPBMC-derived CCP, homed todamaged cardiac tissues, engrafted, and is hypothesized to havetransdifferentiated into cells expressing cardiomyocyte markers.

It is to be noted that ACPs typically improve systemic endothelialfunctioning, as expressed by improved ejection fraction and reducednecrosis. Particular examples of improvement due to administration ofACPs, derived in accordance with an embodiment of the present invention,include improved cardiovascular functioning and improved sexualfunctioning. The scope of the present invention includes identifying apatient having cardiovascular dysfunction or sexual dysfunction, andadministering ACPs to the patient in order to treat the dysfunction.

Example 14

In a production procedure, individual autologous human-PBMC-derived CCPswere cultured in order to generate an ACP-rich PCP, as describedhereinabove. The CCPs were grown on autologous plasma-coated T75 flasksin the presence of medium containing autologous serum (>=10%), 2-10ng/ml VEGF, and 5 IU/ml Heparin. Harvested cells, approved by QualityControl for clinical use, were administrated to patients. Thetherapeutic potential of ACP-rich PCP is summarized in results ofadministration thereof to 14 patients suffering from end-stage heartfailure. Left ventricular ejection fraction (EF) and disease severityscore (Score) were assessed prior to and 1-8 months after the ACP celladministration. Improvement of these parameters was calculated relativeto each patient's baseline evaluation according to the followingequation:

% Improvement=(Test result after treatment−Baseline testresult)/Baseline test result.

Results show statistically significant improvement (p<0.0001; testedusing two-tailed, paired t test analysis) in both parameters followingtreatment by administering ACP-rich PCP.

Table 6 shows the number of treated patients, averages and individualresults relating to EF and disease severity score, as well as thecalculated percent improvement thereof.

TABLE 6 % EF* SCORE* % EF At 1-8 EF % SCORE At 1-8 SCORE % Batch No.Baseline Months Improvement Baseline Months Improvement N 14 14 14 14.014.0 14.0 Average 24.1 34.8 49.8 2.9 1.6 45.6 Range 14.9-36.0 20.0-50.011.1-133 2.0-4.0 1.0-3.0 29.0-67.0 SE 2.1 2.8 10.9 0.1 0.1 4.1 PCEPC06630.0 40.0 33.3 2.00 1.00 50.00 PCEPC081 23.0 50.0 117.4 3.00 1.00 66.67PCEPC083 27.5 32.5 18.2 3.00 2.00 33.33 PCEPC091 14.9 20.0 34.2 3.002.00 33.33 PCEPC092 35.0 41.0 17.1 3.00 2.00 33.33 PCEPC094 36.0 40.011.1 3.00 2.00 33.33 PCEPC097 15.0 27.5 83.3 3.00 1.00 66.67 PCEPC09915.0 35.0 133.3 3.50 2.00 42.86 PCEPC103 18.3 20.9 14.2 3.00 2.00 33.33PCEPC106 15.0 20.0 33.3 3.00 1.00 66.67 PCEPC110 25.0 50.0 100.0 3.002.00 33.33 PCEPC114 22.0 30.0 36.4 2.00 1.00 50.00 PCEPC121 25.0 32.028.0 3.50 2.50 28.57 PCEPC137 35.0 48.0 37.1 3.00 1.00 66.67*Significant improvement p < 0.0001

Example 15

In a separate set of experiments, a human-PBMC-derived CCP was culturedin order to generate a cardiomyocyte (CMC)-rich PCP; the CCP was grownon fibronectin or plasma-coated T75 flasks in accordance with protocolsdescribed herein (see medium preparation).

FIG. 10 is a photograph of a typical CMC-rich PCP from the experimentsof the current example (Example 15), derived in accordance with anembodiment of the present invention. Typically, these cells appearelongated with dark cytoplasm, which may indicate high protein content.This image was obtained from ×200 magnification of cultured CMC-rich PCPcells.

FIGS. 11A, 11B, and 11C are photographs showing immunostaining ofCCP-derived cardiomyocytes in the experiments of the current example(Example 15), in accordance with an embodiment of the present invention.Slide-fixed CMC PCP cells were stained with:

FIG. 11A—anti-cardiac Troponin detected by anti-mouse Cy-3;

FIG. 11B—anti-alpha-actin detected by anti-mouse IgG-FITC; and

FIG. 11C—anti-connexin 43 detected by anti-mouse IgG-FITC.

Cells stained with non-specific mouse IgG were detected by anti-mouseIgG-FITC or by anti-mouse IgG-Cy3 and were used as negative controls.

FIGS. 11A-C show that CMC-rich PCP cells expressed the typicalcardiomyocyte cellular markers: cardiac Troponin T (FIG. 11A),alpha-actin (FIG. 11B), as well as the functionally important GAPjunction marker connexin-43 (FIG. 11C). The images were obtained from×100 magnification of slide-fixed cells.

Example 16

In the same set of experiments that produced the results shown in FIGS.10-11C, a human-PBMC-derived CCP was cultured in order to generate aCMC-rich PCP; the CCP was grown on fibronectin or plasma-coated T75flasks in accordance with protocols described herein (see mediumpreparation).

FIGS. 12A and 12B are graphs showing flow cytometry analysis resultsobtained from immunostaining of a CMC-rich PCP in the experiments of thecurrent example (Example 16), in accordance with respective embodimentsof the present invention. In FIGS. 12A-B, lines describing control,e.g., non-specific staining, are marked as “Control”; specificimmunostaining with the cardiac cellular markers desmin and troponin Tare marked as Desmin (FIG. 12A) and Troponin T (FIG. 12B), respectively.The M1 line represents the statistical marker area in which the cellsare positively stained for the respective marker.

Example 17

In a separate set of experiments, a human-PBMC-derived CCP was culturedin order to generate a CMC-rich PCP, as described hereinabove. TheCMC-rich PCP cells' therapeutic potential was assessed in a rat model ofacute myocardial infarction. CMC-rich PCP cells were used forimplantation into a rat model of induced acute myocardial infarction asdescribed hereinabove with respect to Example 13 (with the exceptionthat CMC-rich PCP cells were used for implantation into the rat model inthe current example, whereas in Example 13, ACP-rich PCP cells were usedfor implantation). Six days after myocardial infarction, heart muscle of9 rats were injected with 1.5×10̂6 CMC PCP cells (CMC, n=9), while heartmuscle of 5 rats were injected with culture medium (Control, n=5).Cardiac function (ejection fraction) was evaluated 14 days following theadministration of the CMC-rich PCP cells or culture medium.

FIG. 13 is a graph showing experimental results obtained in theexperiments of Example 13, in accordance with another embodiment of thepresent invention. It is to be noted that the percentage of ejectionfraction of the CMC-administered rats increased substantially incomparison to the decreased percentage ejection fraction of the controlrats.

A series of protocols are described hereinbelow which may be used, asappropriate, separately or in combination with Examples 1-17, inaccordance with embodiments of the present invention. It is to beappreciated that numerical values are provided by way of illustrationand not limitation. Typically, but not necessarily, protocols may bederived using values selected from a range of values that is within 20%of the value shown. Similarly, although certain steps are describedherein with a high level of specificity, a person of ordinary skill inthe art will appreciate that additional or other steps may be performed,mutatis mutandis.

In accordance with an embodiment of the present invention; generation ofa single-cell suspension is carried out using the following protocols:

Protocol 1: Extraction of Peripheral Blood Mononuclear Cells (PBMC).

-   -   Receive blood bag and sterilize it with 70% alcohol. Load blood        cells onto a Ficoll™ gradient.    -   Spin the tubes for 20 minutes at 1050 g at room temperature        (RT), with no brake.    -   Collect most of the plasma from the supernatant.    -   Collect the white blood cell fraction from every tube.    -   Transfer the collected cells to a new 50 ml tube, adjust volume        to 30 ml per tube using PBS.    -   Spin tubes for 15 minutes at 580 g, RT, and discard supernatant.    -   Count cells in Trypan Blue.    -   Re-suspend in culture medium comprising, for example, X-vivo        15™.        Protocol 2: Extraction of Cells from Umbilical Cord.    -   Isolate 10 cm umbilical cord.    -   Wash thoroughly with sterile PBS.    -   Identify the major vein of the cord, and clamp one end of the        vein.    -   Wash twice with 30 ml sterile PBS.    -   Fill vein with 0.15% collagenase (about 5 ml of 0.15%        collagenase solution).    -   Clamp the second end of the vein.    -   Incubate at 37 C for 15 min.    -   Wash outer side of the cord with 70% ethanol.    -   Release the clamp from one end of the vein and collect the cell        suspension.    -   Centrifuge for 10 min at 580 g, 21 C.    -   Re-suspend the cells in culture medium comprising, for example,        X-vivo 15™, 10% autologous serum, 5 IU/ml heparin, and one or        more growth factors.        Protocol 3: Extraction of Cells from Bone Marrow.    -   Get bone marrow aspiration from surgical room.    -   Re-suspend in culture medium comprising, for example, X-vivo        15™, 10% autologous serum, 5 IU/ml heparin, and one or more        growth factors.    -   Pass suspension through a 200 um mesh.

In accordance with an embodiment of the present invention, generation ofa CCP is carried out using the following protocols:

Protocol 1: Generation of a Human CCP from PBMCs Using a Percoll™Gradient.

-   -   Prepare gradient by mixing a ratio of 5.55 Percoll™ (1.13 g/ml):        3.6 ddH2O: 1 PBSx10.    -   For every 50 ml tube of Percoll: mix 20 ml of Percoll™ stock, 13        ml of ddH2O and 3.6 ml of PBSx10.    -   Mix vigorously, by vortexing, for at least 1 min.    -   Load 34 ml mix into each 50 ml tube.    -   Centrifuge tubes, in a fixed angle rotor, for 30 min at 17,000        g, 21 C, with no brake.    -   Gently layer 3.0 ml of cell suspension of 150 million-400        million PBMCs on top of the gradient.    -   Prepare a second tube with density marker beads: gently layer        3.0 ml of medium on top of the gradient.    -   Gently load density marker beads—10 ul from each bead type.

Centrifuge tubes, in a swinging bucket rotor, for 30 min at 1260 g at 13C, with no brake.

-   -   Gently collect all bands located above the red beads, and        transfer to tube with 10 ml medium.    -   Centrifuge cells for 15 min at 580 g at 21 C.    -   Discard supernatant and re-suspend pellet in medium.    -   Count cells in Trypan blue.    -   Centrifuge cells for 10 min at 390 g, 21 C.    -   Discard supernatant and re-suspend pellet in medium.    -   Take CCP cells for FACS staining.        Protocol 2: Generation of Human CCP from PBMCs Using an        OptiPrep™ Gradient.    -   Take up to 130 million cells for each enrichment tube.    -   Spin cells for 10 min at 394 g, 21 C.    -   Suspend cell pellet in 10 ml of donor serum.    -   Prepare a 1.068 g/ml OptiPrep™ gradient by mixing a ratio of 1        OptiPrep™: 4.1 PBS.    -   For every 50 ml enrichment tube:    -   Mix 10 ml of cell suspension with 4 ml OptiPrep™.    -   For preparation of a 1.068 g/ml OptiPrep™ gradient, mix 5 ml of        OptiPrep™ and 20.5 ml of PBS.    -   Gently layer 20 ml of the 1.068 g/ml gradient on top of the cell        suspension.    -   Gently layer 1.5 ml Hank's buffered saline (HBS) on top of the        gradient layer.    -   Centrifuge for 30 min at 700 g at 4 C, with no brake.    -   Gently collect the layer of cells that floats to the top of the        1.068 g/ml OptiPrep™ gradient into a 50 ml tube pre-filled with        PBS.    -   Centrifuge for 10 min at 394 g, 21 C.    -   Discard supernatant and re-suspend pellet in medium.    -   Count cells in Trypan Blue.

It is to be noted that culture containers are typically either un-coatedor coated with one or a combination of ACP-enhancing materials such ascollagen, fibronectin, CD34, CD133, Tie-2, or anti-CD117.

In accordance with an embodiment of the present invention, the coatingof a tissue culture container is carried out using the followingprotocols:

Protocol 1: Coating T75 Glasks with 25 ug/ml Fibronectin.

-   -   For 20 T75 flasks—Prepare up to seven days before, or on day of        PBMC preparation.    -   Prepare 50 ml of 25 ug/ml fibronectin solution in PBS.    -   Fill every flask with 2-5 ml fibronectin 25 ug/ml.    -   Incubate at 37 C for at least 30 min.    -   Collect fibronectin solution.    -   Wash flask twice in PBS.    -   Dry flasks    -   Keep dry flasks at room temperature.    -   Dried flasks can be saved for one week at room temperature (RT).        Protocol 2: Coating T75 Flasks with 25 ug/ml Fibronectin and 5        ng/ml BDNF    -   Coat flasks with Fibronectin 25 μg/ml, as described in Protocol        1.    -   Prepare 50 ml of 5 ng/ml BDNF solution in PBS.    -   After washing off Fibronectin, fill every flask with 2-5 ml BDNF        10 ng/ml.    -   Incubate at 37 C for 1 hour.    -   Collect the solution.    -   Wash flask twice in 10 ml PBS.    -   Keep dry flasks at room temperature until use.

In accordance with an embodiment of the present invention, serumpreparation is carried out using the following protocol: (Serum can beobtained directly or prepared from plasma).

Protocol: Preparation of serum from human plasma.

-   -   Take 100 ml of undiluted blood.    -   Spin at 1100 g (2500 rpm) for 10 min.    -   Transfer the upper layer (plasma) to a new 50 ml tube.    -   Add 1.0 ml 0.8M CaCl₂-2H₂O for every 40 ml plasma.    -   Incubate for 0.5-3 hours at 37 C.    -   Spin coagulated plasma 5 min at 2500 g.    -   Collect the serum in a new tube, avoiding clotting.    -   Aliquot collected serum and save at −20 C until use.

In accordance with an embodiment of the present invention, mediumpreparation is carried out using the following protocols:

Medium should contain 1-20% autologous serum and/or 1-20% conditionedmedium.

Medium can contain one or more additives, such as LIF, EPO, IGF, b-FGF,M-CSF, GM-CSF, TGF alpha, TGF beta, VEGF, BHA, BDNF, NGF, EGF, NT3,NT4/5, GDNF, S-100, CNTF, NGF3, CFN, ADMIF, estrogen, progesterone,cortisone, cortisol, dexamethasone, or any other molecule from thesteroid family, prolactin, an adrenocorticoid hormone, ACTH, glutamate,serotonin, acetylcholine, NO, retinoic acid (RA) or any other vitamin Dderivative, Heparin, insulin, forskolin, Simvastatin, MCDB-201, MCT-165,glatiramer acetate, a glatiramer acetate-like molecule, IFN alpha, IFNbeta or any other immunoregulatory agent sodium selenite, linoleic acid,ascorbic acid, transferrin, 5-azacytidine, PDGF, VEGF, cardiotrophin,and thrombin or Rosiglitazone in various concentrations, typicallyranging from about 100 pg/ml to about 100 μg/ml (or molar equivalents).

Typically, medium should not be used more than 10 days from itspreparation date.

Protocol 1: Medium for Enhancement of CCP-Derived Angiogenic CellPrecursors (ACPs).

-   -   Serum-free medium (e.g., X-vivo 15™)    -   10% autologous serum    -   5 IU/ml Heparin    -   5 ng/ml VEGF    -   1 ng/ml EPO

Protocol 2: Medium for Enhancement of CCP-Derived Neuronal ProgenitorCells.

-   -   Serum-free medium (e.g., X-vivo 15™)    -   20 ng/ml bFGF    -   50 ng/ml NGF    -   200 uM BHA (this is added during the last 24 hours of culturing)    -   10 ng/ml IFN beta    -   10 ug/ml glatiramer acetate    -   10 uM forskolin    -   1 uM cortisone    -   1 ug/ml insulin

Protocol 2.1: Medium for Enhancement of CCP-Derived Neuronal ProgenitorCells.

-   -   Serum-free medium (e.g., X-vivo 15™)    -   20 ng/ml bFGF    -   50 ng/ml NGF    -   25 ng/ml BDNF    -   200 uM BHA (this is added during the last 24 hours of culturing)

Protocol 3: Medium for Enhancement of CCP-Derived Retinal Cells.

-   -   Serum-free medium (e.g., X-vivo 15™)    -   10% autologous serum    -   5 IU/ml Heparin    -   10 ng/ml EGF    -   20 ng/ml bFGF    -   10 ug/ml glatiramer acetate    -   50 ng/ml NGF3        Protocol 4a. Medium for Enhancement of CCP-Derived        Cardiomyocyte (CMC) Progenitor Cells.

Step I

-   -   Serum-free medium (e.g., X-vivo 15™)    -   10% autologous serum    -   20 ng/ml bFGF    -   20 ug/ml IFN beta    -   5 IU heparin.

Step II

-   -   Five to ten days after culture onset, add 3 uM 5-azacytidine for        24 hours.

Protocol 4b: Medium for Enhancement of CCP-Derived CMC Progenitor Cells.

-   -   Serum free medium DMEM-Low glucose    -   20% autologous serum    -   10% MCDB-201    -   2 ug/ml Insulin    -   2 ug/ml Transferin    -   10 ng/ml Sodium Selenite    -   50 mg/ml BSA    -   1 nM Dexamethasone    -   20 ug/ml Glatiramer acetate    -   0.47 ug/ml Linoleic acid    -   0.1 mM Ascorbic Acid    -   100 U/ml penicillin

In accordance with an embodiment of the present invention, conditionedmedium preparation is carried out using the following protocol:

Protocol 1: Preparation of 100 ml Enriched Medium Containing 10%Autologous Conditioned Medium.

-   -   Thaw 10 ml conditioned medium in an incubator.    -   When thawed, add it to culture medium using pipette.

Extraction of Tissue Pieces for Co-Culture:

Dissection of rat blood vessels (other non-human or human tissues mayalso be used):

-   -   Anesthetize animal using anesthetic reagents (e.g., 60-70% CO2,        isoflurane, benzocaine, etc.).    -   Lay animal on its back and fix it to an operating table.    -   Using sterile scissors, cut animal's skin and expose the inner        dermis.    -   Using a second set of sterile scissors, cut the dermis, cut        chest bones, and expose the heart and aorta.    -   Cut small pieces, 0.2 - 1 cm long, from the aorta and other        blood vessels, and place them in a container pre-filled with 50        ml cold culture medium (e.g. RPMI, X-vivo 15™), or any other        growth medium).    -   Using forceps and scissors, clean tissue sections, to remove        outer layers such as muscle, fat, and connective tissue.    -   Using forceps and scalpel, cut each blood vessel along its        length, and expose the inner layer of endothelial cells.    -   Using forceps and scalpel, cut small pieces of up to 0.1 cm2        from the tissue.

It is to be understood that whereas this technique is in accordance withone embodiment of the present invention, the scope of the presentinvention includes extracting a blood vessel from a human, as well. Forexample, an incision may be made over the saphenous vein, in order tofacilitate dissection of a distal 1 cm portion of the vein. Tributaryveins thereto are tied and transected. Distal and proximal ends of the 1cm portion of the saphenous vein are tied, and the vein is harvested.

Use the dissected tissue for direct and/or indirect co-culturing withthe CCP and/or to generate conditioned medium.

Generation of Conditioned Medium:

-   -   Lay dissected pieces in culture containers, for example in T75        flasks, or 50 ml tubes.    -   Optionally, fill with cell culture medium containing 0.1-3 ug/ml        or 3-100 ug/ml apoptotic reagent (such as valinomycin, etoposide        or Staurosporine), until all pieces are covered.    -   Refresh culture medium every 2 days.    -   Collect this medium (now conditioned medium) into 50 ml tubes.    -   Spin collected conditioned medium at 450 g for 10 min, at room        temperature.    -   Collect supernatant in a new sterile container.

Details regarding preservation of the conditioned medium, in accordancewith an embodiment of the present invention, are described hereinbelow.

In accordance with an embodiment of the present invention, culturing ofa CCP to produce a PCP is carried out using the following protocols:

Protocol 1: Culturing of CCP Cell Suspension in T75 Flasks.

-   -   Spin suspension for 15 minutes at 450 g, 21 C.    -   Discard the supernatant.    -   Gently, mix cell pellet and re-suspend the CCP cells.    -   Re-suspend pellet to 10 million CCP cells/ml.    -   Fill T75 flask with 15 ml enriched medium, and add 5 ml of 10        million CCP cells/ml to attain a final concentration of 50        million CCP cells/flask.    -   Incubate T75 flasks, plates and slides at 37 C, 5% CO2.

Protocol 2: Applied Hypoxia.

For some applications, increased expansion and/or differentiation of theCCP may be obtained by exposure of the cell culture to oxygenstarvation, e.g., 0.1-5% or 5-15% oxygen (hypoxia), for 2-12 or 12-48hours. This is typically done one or more times, at different pointsduring cell culturing.

Incubate T75 flasks in an oxygen-controlled incubator.

Set the oxygen pressure at 0.1%, and maintain it at this level for 24hours.

Remove the flasks from the incubator and examine the culture.

Take a sample of CCP cells and test viability by Trypan blue exclusionmethod.

Set the oxygen pressure of the incubator at 20%.

-   -   Re-insert the flasks into the incubator and continue incubation        for the rest of the period. This procedure can be repeated, for        example, once a week during the culture period and/or within 24,        48, or 72 hours before termination of the culture.        Protocol 3: Reseeding of Adherent and/or Detached and/or        Floating Cells.

For some applications, increased expansion and differentiation of theCCP may be achieved by re-seeding collected cells on new pre-coateddishes in culture medium.

-   -   Collect all cultured CCP in tubes.    -   Spin tubes for 10 minutes at 450 g, 21 C.    -   Discard the supernatant.    -   Gently mix pellet and re-suspend cells in 10 ml fresh medium per        T75 flask.    -   Seed suspended cells in new pre-coated T75 flasks.    -   Continue culturing the cells, and perform all other activities        (e.g., medium refreshment, visual inspection, and/or flow        cytometry), as appropriate, as described herein.

This procedure can be performed weekly during the culture period and/orwithin 24, 48, or 72 hours before termination of the culture.

In accordance with an embodiment of the present invention, co-culturingof

CCP with tissue-derived conditioned medium is carried out using thefollowing protocol:

Protocol 1: Culturing of CCP in the Presence of Conditioned MediumDerived from a Blood Vessel Culture.

-   -   Spin CCP cells for 15 minutes at 500 g, 21 C.    -   Discard the supernatant.    -   Gently mix cell pellet and re-suspend cells to 5-50 million/ml        in autologous medium containing 1-20% autologous serum and/or        1-20% conditioned medium.    -   Seed flasks with 2-5 million CCP cells/ml.    -   Incubate flasks at 37 C, 5% CO2.    -   After first three days of culture, non-adherent cells can be        removed from the culture.

In accordance with an embodiment of the present invention, refreshing ofthe media in ongoing growing CCP cultures is carried out using thefollowing protocol:

Refreshing of the media in ongoing growing flasks should occur every 3-4days.

Protocol 1: Refreshing of Medium in T-75 Flasks.

-   -   Collect non-adherent cells in 50 ml tubes.    -   Fill every flask with 10 ml fresh culture medium enriched with        conditioned medium.    -   Spin tubes for 10 minutes at 450 g, RT; discard the supernatant.    -   Gently mix cell pellet and re-suspend cells in 10 ml/flask fresh        culture medium enriched with condition medium.    -   Return 5 ml of cell suspension to every flask.

In accordance with an embodiment of the present invention, indirectco-culture of CCP cells with tissue dissection is carried out using thefollowing protocol:

Protocol 1: Indirect Co-Culture of Dissected Blood Vessel and CCP Cellsin a Semi-Permeable Membrane Apparatus.

-   -   Lay dissected tissue pieces in the upper chamber of the        apparatus on top of the semi-permeable membrane.    -   Implant CCP cells in lower chamber.    -   Lower chamber can be pre-coated with growth-enhancing molecules        such as collagen, plasma, fibronectin, a growth factor,        tissue-derived extra cellular matrix and an antibody.    -   Refresh culture medium in the upper chamber—aspirate conditioned        medium into 50 ml tubes and add autologous culture medium.    -   Preserve collected conditioned medium at −20 C.    -   Remove upper chamber after four days of co-culture.    -   Refresh culture medium of the CCP cells with culture medium        containing 1-20% autologous serum and/or 1-20% conditioned        medium.    -   Continue growing and harvesting as described herein.    -   Co-culture in separate chambers within a culture container

In accordance with an embodiment of the present invention, co-culturingwithin a culture container is carried out using the following protocol:

Protocol 1: Direct Co-Culturing of Autologous Dissected Blood Vessel andCCP Cells.

-   -   Lay dissected tissue pieces in pre-coated flasks.    -   Implant CCP cells in pre coated second chamber.    -   Using forceps, take out tissue pieces after four days of        co-culture.    -   Refresh culture medium of the CCP cells with culture medium        containing 1-20% autologous serum and/or 1-20% condition medium.    -   Continue growing and harvesting as described herein.

In accordance with an embodiment of the present invention, harvesting ofthe cellular product is carried out using the following protocol:

Protocol 1: Collection of Resulting ACP Cultures.

-   -   Collect cells in 50 ml tubes.    -   Carefully wash flask surface by pipetting with cold PBS to        detach adherent cells.    -   Collect washed adherent cells to 50 ml tubes.    -   Add 5 ml of cold PBS.    -   Detach remaining adherent cells using gentle movements with cell        scraper.    -   Collect the detached cells and add them to the tubes    -   Optionally, add 5 ml EDTA to each flask and incubate at 37 C for        5 min.    -   Collect the detached cells and add them to the tubes Spin tubes        for 5 min, at 450 g, room temperature.    -   Re-suspend the pellets in 2-5 ml PBS.    -   Count the cells in Trypan blue.

In accordance with an embodiment of the present invention, cellularproduct preservation is carried out using the following protocols:

Cellular product can be kept in preservation media or frozen in freezingbuffer until use for transplantation into a patient.

Protocol 1: Cryopreservation of Cellular Product.

-   -   Prepare freezing buffer containing 90% human autologous serum        and 10% DMSO.    -   Suspend cellular product in freezing buffer and freeze in liquid        nitrogen.

Protocol 2: Short-Period Preservation of Cellular Product.

Prepare preservation medium including growth medium containing 1-20%autologous serum, with few or no other additives. Maintain preservationmedium with cellular product at 2-12 C

In accordance with an embodiment of the present invention, conditionedmedium collection and preservation is carried out using the followingprotocol:

-   -   Conditioned medium can be kept until use for growth medium        preparation.    -   Conditioned medium should be collected under sterile conditions.    -   Spin collected conditioned medium for 10 min at 450 g, 21 C.    -   Collect supernatant in a new sterile container.    -   Filter supernatant through a 22 um membrane.    -   Aliquot conditioned medium to 10 and/or 50 ml sterile tubes,        pre-marked with donor details.

Keep at −20 C until use.

In accordance with an embodiment of the present invention, FACS stainingis carried out using the following protocol:

Protocol 1: Staining of ACP Enriched Population.

FACS staining protocol:

Tube No. Staining Aim of staining 1. Cells Un-stained control 2. CD45(IgG1)-FITC Single staining for PMT and 3. CD14-PE (IgG2a) compensationsettings 4. CD45 (IgG1)-APC 5. mIgG1-FITC Isotype control mIgG1-PEmIgG1-APC 6. CD45-FITC (IgG1) KDR-PE (IgG2a) CD34-APC (IgG1) 7.Ac-LDL-FITC CD31-PE (IgG1) 8. Ulex-Lectin-FITC CD31-PE (IgG1) 9.mIgG1-FITC Isotype control mIgG2a-PE mIgG1-APC 10. CD45-FITC (IgG1)CD133-PE (IgG2a) CD34-APC (IgG1)

Protocol 2: Staining of CMC Progenitors.

FACS staining protocol for fixed permeabilized cells:

Staining Staining Tube No. 1^(st) step 2^(nd) step Aim of staining 1Cells Un-stained control 2 CD45-FITC (IgG1) Single staining for 3CD14-PE (IgG2a) PMT and compensation settings 5 mIgG1 Anti mouse-PEIsotype control 6 Desmin Anti mouse-PE 7 Troponin T Anti mouse-PEIsotype control

In accordance with an embodiment of the present invention,immunohistochemistry staining (IHC) is carried out using the followingprotocols:

Protocol 1: IHC Staining Protocol for ACPs.

Slide Staining No. 1st step Aim of staining 1. mIgG1 Isotype control 2.mIgG1-PE Isotype control 3. CD34-APC Specific Staining 4. CD144-FITCSpecific Staining 5. CD133-PE Specific Staining 6. Ac-LDL-FITC SpecificStaining CD31-PE 7. Ulex-Lectin-FITC Specific Staining CD31-PE

Protocol 2: IHC Staining Protocol for CMC Progenitors.

Slide Staining Staining No. 1st step 2nd step Aim of staining 1. —mIgG1-FITC Isotype control 2. mIgG1 Anti mouse-Cy-3 Isotype control 3.Connexin 43 Anti mouse-FITC Specific Staining 4. Alfa actin Antimouse-FITC Specific Staining 5. Troponin Anti mouse-PE Specific Staining

In accordance with an embodiment of the present invention, a tubeformation assay is carried out using the following protocol:

Tube formation was tested using the ECM625(Chemicon) in vitroangiogenesis assay kit.

Angiogenic pattern and vascular tube formation was numerically scored asdescribed by Kayisli U.A. et al. 2005 (52).

In accordance with an embodiment of the present invention, secretion ofcytokines from harvested cells is assessed using the followingprotocols:

-   -   Culture 0.5-1×10̂6 cells/ml over night in 24 well plates in        serum-free medium (e.g., X-vivo 15)    -   Collect culture supernatant and spin at 1400 rpm for 5 minutes    -   Transfer supernatant to an eppendorf tube and freeze at −80 C        until ready to test cytokine secretion.

Protocol 1: ELISA for IL-8.

-   -   A commercial DuoSet CXCr8/IL-8 (R&D Systems) was used for the        detection of IL-8 secretion.

Protocol 2: Cytometric Bead Array.

-   -   A commercial cytometric bead array (CBA) kit for human        angiogenesis (BD 558014) was used for the detection of IL-8,        VEGF, TNF and Angiogenin secretion.

It is to be noted that the scope of the present invention includesinjecting IL-8 into a human patient in order to recruit ACP cells to agiven destination within a given patient, in accordance with the needsof the patient.

For some applications, techniques described herein are practiced incombination with techniques described in one or more of the referencescited in the Background section and Cross-References section of thepresent patent application. All references cited herein, includingpatents, patent applications, and articles, are incorporated herein byreference.

It is to be appreciated that by way of illustration and not limitation,techniques are described herein with respect to cells derived from ananimal source. The scope of the present invention includes performingthe techniques described herein using a CCP derived from non-animalcells (e.g., plant cells), mutatis mutandis.

It will be appreciated by persons skilled in the art that the presentinvention is not limited to what has been particularly shown anddescribed hereinabove. Rather, the scope of the present inventionincludes both combinations and subcombinations of the various featuresdescribed hereinabove, as well as variations and modifications thereofthat are not in the prior art, which would occur to persons skilled inthe art upon reading the foregoing description.

1-14. (canceled)
 15. A composition of matter, comprising a population ofcultured cells that comprises a sub-population of cells that stain asCD31Bright, demonstrate uptake of Ac-LDL+ and secrete interleukin-8. 16.The composition of matter according to claim 15, wherein thesub-population comprises at least 10% of the cells in the population.17-18. (canceled)
 19. The composition of matter according to claim 15,wherein the sub-population secretes at least 50 pg IL-8 per 10⁶ cells/mlover a period of at least 24 hours. 20-159. (canceled)
 160. Thecomposition of matter according to claim 15, wherein at least 1.5% ofthe cells of the population comprise a feature selected from the groupconsisting of: CD34, CD117, CD133, Tie-2, CD34+CD133+, KDR, CD34+KDR+,CD144, von Willebrand Factor, SH2 (CD105), SH3, fibronectin, collagentype I, collagen type III, collagen type IV, ICAM type 1, ICAM type 2,VCAM1, vimentin, BMP-R IA, BMP-RII, CD44, integrin b1, aSM-actin, MUC18,CXCR4 and CXCR8.
 161. The composition of matter according to claim 15,wherein at least 1.5% of the cells of the population secrete a moleculeselected from the group consisting of: angiogenin, VEGF, MMP2, and MMP9.162. The composition of matter according to claim 15, wherein at least1.5% of the cells of the population include a feature selected from thegroup consisting of: a tube-like structure, a tendency to form a colony,a tendency to form a cluster, and a tendency to migrate toward achemoattractant selected from the group consisting of: bFGF, VEGF, SCF,G-CSF, GM-CSF, SDF-1, and IL-8.
 163. A method comprising: providing aninitiating cell population (ICP) of at least 5 million cells that have adensity of less than 1.072 g/ml, at least 1% of which areCD34+CD45−/Dim, and at least 25% of which are CD31Bright; andstimulating the ICP to differentiate into a progenitor/precursor cellpopulation (PCP).
 164. The method according to claim 163, wherein theICP includes at least 5 million cells, and wherein stimulating the ICPcomprises stimulating the ICP that includes the at least 5 millioncells.
 165. The method according to claim 163, wherein at least 1.5% ofthe cells of the ICP are CD34+CD45−/Dim, and wherein stimulating the ICPcomprises stimulating the ICP of which at least 1.5% of the cells areCD34+CD45−/Dim.
 166. The method according to claim 163, wherein at least30% of the cells of the ICP are CD31Bright, and wherein stimulating theICP comprises stimulating the ICP of which at least 30% of the cells areCD31Bright.
 167. The method according to claim 163, wherein the ICPincludes at least 5 million cells that have a density of less than 1.062g/ml, at least 1% of which are CD34+CD45−/Dim, and wherein stimulatingthe ICP comprises stimulating the ICP that has the at least 5 millioncells that have a density of less than 1.062 g/ml.
 168. The methodaccording to claim 163, further comprising preparing the PCP as aproduct for administration to a patient.
 169. The method according toclaim 163, wherein the ICP is characterized by at least 2.5% of the ICPbeing CD34+CD45−/Dim, and wherein stimulating the ICP comprisesstimulating the ICP having the at least 2.5% of the ICP that areCD34+CD45−/Dim.
 170. The method according to claim 163, whereinstimulating the ICP comprises culturing the ICP for a period lastingbetween 1 and 5 days in a culture medium comprising less than or equalto 10% serum.
 171. The method according claim 163, wherein stimulatingthe ICP comprises culturing the ICP in the presence of IL-8.
 172. Themethod according to claim 163, further comprising identifying the PCP asbeing suitable for therapeutic implantation in response to an assessmentthat at least 1.5% of cells of the PCP demonstrate a feature selectedfrom the group consisting of: a desired morphology, a desired cellularmarker, a desired cellular component, a desired enzyme, a desiredreceptor, a desired genotypic feature, and a desired physiologicalfeature.
 173. The method according claim 163, further comprisingidentifying the PCP as being suitable for therapeutic implantation inresponse to an assessment that the PCP includes at least 1 millionangiogenic cell precursors (ACPs).
 174. The method according to claim163, further comprising identifying the PCP as being suitable fortherapeutic implantation in response to an assessment that the PCPincludes at least 1 million cardiomyocyte progenitors.
 175. The methodaccording to claim 163, further comprising identifying the PCP as beingsuitable for therapeutic implantation in response to an assessment thatthe PCP includes at least 1 million neural cell progenitors.
 176. Themethod according claim 163, further comprising characterizing the PCP asincluding angiogenic cell precursors (ACPs), in response to anevaluation of at least one feature selected from the group consistingof: a phenotypical feature of cells in the PCP, a genotypical feature ofcells in the PCP, and a physiological feature of cells in the PCP.